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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
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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. The dishes were placed to 25 C and 12:12 h (light: from ancestors close to the wood feeding cockroaches (Lo darkness) photoperiodic regime for one week before the ter- et al., 2000). The fossil records document that termites have mites were sacrificed in the middle of the photophase. Three not changed their over-all morphology and apparently also species were not exposed to these controlled conditions. Hodo- not their eusocial life style for 130 million years (Thorne termes mossambicus and Macrotermes jeanneli were taken et al., 2000). Phenotypic diversification of castes (certain from old colonies maintained in the Zoological Garden of castes are derived only from one sex in some species) is a char- Bern (Switzerland) under natural light regime. Termites were acteristic feature of their colonies that typically include a royal collected in the mid-day and immediately fixed. Odontotermes reproductive pair (mature colonies usually contain also sec- feae was collected outdoors in North-East Thailand in the mid- ondary reproductives), larvae (immature individuals without dle of the day and fixed on the spot. Author's personal copy
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Table 1 List of examined species and castes (workers and pseudergates exclude each other) Species Subfamily Family Caste used Mastotermes darwiniensis (Froggat) e Mastotermitidae Workers Zootermopsis angusticollis (Hagen) e Termopsidae Pseudergates Hodotermes mossambicus (Hagen) e Hodotermitidae Workers Neotermes castaneus (Burmeister) e Kalotermitidae Pseudergates Prorhinotermes simplex (Hagen) Prorhinotermitinae Rhinotermitidae Pseudergates Reticulitermes flavipes (Kollar)* Heterotermitinae Rhinotermitidae Workers Coptotermes formosanus Shiraki Coptotermitinae Rhinotermitidae Workers Macrotermes jeanneli (Grasse´) Macrotermitinae Termitidae Workers Odontotermes feae (Wasmann) Macrotermitinae Termitidae Workers Nasutitermes costalis (Holmgren) Nasutitermitinae Termitidae Workers *Introduced into Europe and reported as R. santonensis Feytaud (Cle´ment et al., 2001).
2.2. Primary antibodies The sections were rinsed three times with PBST and incu- bated for 1 h with the goat anti-rabbit IgG secondary antibody Rabbit polyclonal antiserum against [His7]-corazonin conjugated to horseradish peroxidase (Jackson ImmunoRe- (Crz, pETFQYSHGWTNa) was prepared by Wako Co., Na- search, diluted 1:1000 in PBST). Following three washes gano, Japan, and made available to us by Dr. Seiji Tanaka. (10 min each) in PBST and one in 0.05 M TriseHCl (pH Antiserum reactivity to [Arg7]-Crz was demonstrated by 7.4, 10 min), the peroxidase activity was visualised with hy- Roller et al. (2003). The antiserum against synthetic PDH drogen peroxide (0.005%) and 3,30-diaminobenzidine tetrahy- of crustaceans (NSELINSILGLPKVMNDAa) was raised in drochloride (0.25 mM in 0.05 M TriseHCl, pH 7.4). Stained rabbits and characterised by Dr. H. Dircksen (Dircksen sections were dehydrated and mounted in DPX medium et al., 1987), from whom it was obtained for our study. (Fluka). Helfrich-Fo¨rster and Homberg (1993) demonstrated that the 8 15 antibody recognised [Leu , Leu ]-PDF in the nervous sys- 2.4. Whole mount immunocytochemistry tem of fruit flies. The specificity of immunoreactions in our preparations was verified by replacing primary antibody Dissected complexes Br-SOG were fixed in 4% paraformal- with a non-immune serum; no immunostaining was detected dehyde in PBS, pH 7.4, at 4 C overnight. After a transfer to in these tests. RT the organs were rinsed in PBST, bathed in collagenase (Sigma; 0.5 mg/ml in PBS) for 30e60 min, washed with 2.3. Immunocytochemistry in paraffin sections methanol and PBST, and treated for 2 h with normal goat se- rum (10% in PBST). Then they were incubated with a primary The brain-suboesophageal ganglion (Br-SOG) complexes antibody for 48 h at 4 C, washed thoroughly with PBST were either dissected in saline (7.5 g NaCl, 0.1 g KCl, 0.2 g (three times 10 min), and incubated overnight at 4 C with CaCl2.2 H2O, and 0.2 g NaHCO3 per 1 L distilled water) a secondary goat anti-rabbit antibody conjugated to horserad- and kept overnight at 4 C in modified BouineHollande fixa- ish peroxidase. The incubation was terminated with three tive (Watson et al., 1993) or the whole head (mouth parts were washes (10e30 min each) in PBST. The peroxidase activity cut off) was submerged in the fixative before dissection of the was detected as described above and the preparations were nervous complex. Fixation in Bouin solution at room temper- mounted in 80% glycerol. ature (RT) was tested in R. flavipes. Heads were left in this fix- ative for 1, 3, and 8 days, respectively, without any effect on 2.5. Data collection and presentation the staining intensity. Based on this experience, Bouin solution was used for the fixation and shipment of the heads of H. mos- The distribution of immunoreactivities in Br-SOG was sambicus, M. jeanneli, and O. feae. first examined in whole mounts under a dissection micro- Fixed Br-SOG were dehydrated through an ethanol series scope. Drawings were prepared based on 6e8 preparations and chloroform and embedded in paraplast. Sections 4e and selected whole mounts were photographed with an 10 mm thick were brought to water and those fixed in Olympus CCD camera and SZX 12 microscope. A Zeiss BouineHolland fixative were treated with Lugol’s iodine Axioplane 2 microscope equipped with Nomarski optics and 5% sodium thiosulfate to remove residual heavy metals. and CCD camera was used to view and photograph the sec- Thoroughly washed sections were transferred through phos- tions. Immunostaining was tracked through the whole gan- phate-buffered saline supplemented with 0.3% Tween 20 glia in complete section series. The studies of whole (PBST) to 10% normal goat serum in PBST. After 30 min mounts and sections complemented each other. Unless men- they were transferred to a humidified chamber and treated tioned otherwise, the numbers of immunoreactive cells are overnight at 4 C with the primary antibodies diluted given per half of the bilaterally symmetrical Br-SOG 1:10,000 with PBST. Further processing was done at RT. complex. Author's personal copy
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3. Results suboesophageal ganglion (SOG) that harboured on each side 2 large Crz-ir perikarya (Fig. 1Dc) located dorsally in the man- 3.1. Corazonin-immunoreactivity (Crz-ir) dibular neuromere, and 2 parikarya of uneven size in the ventral region of the labial neuromere. Four to 6 small and weekly Mastotermes darwiniensis, Zootermopsis angusticollis, and stained cells were located centrally in the maxillary neuromere Hodotermes mossambicus, which represented the families (Fig. 1D). The SOG further contained gentle fibre networks Mastotermitidae, Termopsidae, and Hodotermitidae, respec- along trajectories connecting the brain with the ventral nerve tively, contained in the fronto-lateral protocerebrum two triads cord. The fibres of these networks probably originated in the of Crz-ir cells (Fig. 1Aa,Ab,Ba,Bb,Ca). The fibres of these brain. cells formed a network in the ipsilateral protocerebrum ven- The Crz-ir system of Prorhinotermes simplex (Fig. 1E) trally to the Crz-ir cells and extended to the opposite brain (Rhinotermitidae: Prorhinotermitinae) included 8 perikarya ar- hemisphere through 2 or 3 routes. One of them, which was ranged in three groups in the fronto-lateral protocerebrum absent in Z. angusticollis, was represented by multiple fibres (Fig. 1Ea). The most dorsal and the medial group each con- transversing the central body; in H. mossambicus, some of sisted of 3 cells (Fig. 1Eb), and the ventral group of 2 cells. these fibres dispersed in an elaborate network with varicosi- Fibres emanating from all three groups arborized in a dense ties. Two routes connecting brain hemispheres were alike in and strongly stained network that occupied the frontal part all three species (Fig. 1AeC): a transverse tract passing of protocerebrum around the mushroom body. Several fibres through the protocerebral bridge and another tract extending provided a connection to the opposite hemisphere through initially in fronto-dorsal direction but looping back above the central body. No extensions to other parts of the brain the mushroom body and running ventrally to the base of pro- were detected. A loose bundle of Crz-ir fibres in the SOG con- tocerebrum. Two fine fibres separated from this tract and nected ventral nerve cord with the circumoesophageal connec- passed through the deutocerebrum and tritocerebrum to the tive; it probably supplied a weakly stained fibre running frontal ganglion (FG) in H. mossambicus. At the base of the through the frontal nerve and ramifying in the FG. The peri- protocerebrum, the tract connected opposite brain hemispheres karya of these fibres were not identified, similar to the well in M. darwiniensis and Z. angusticollis, whereas in H. mossam- stained fibres that dispersed in the frontal CC region and pro- bicus it innervated the ipsilateral corpus cardiacum (CC) jected to the ventral part of the CA in P. simplex. (Fig. 1Cb). Crz-ir fibres were present in the nervi corporis car- Representatives of other subfamilies of Rhinotermitidae, diaci and ramified in the CC of all species but connection of Reticulitermes flavipes (Fig. 2A) (Heterotermitinae) and Cop- these fibres to the protocerebral Crz-ir system could be traced totermes formosanus (Fig. 2B) (Coptotermitinae) possessed only in H. mossambicus. Most fibres terminated in the fronto- two triads of Crz-ir somata in the lateral protocerebrum dorsal part of CC in M. darwiniensis, in their caudal region in (Fig. 2Aa,Ba), 4 cells in PI (Fig. 2Bb), 2 cells in the deuto- Z. angusticollis, and continued to the frontal part of corpora cerebrum, and 2 pairs of cells in the frontal part of the SOG allata (CA) in H. mossambicus. (Fig. 2Ab). Fibres from the lateral cells followed three The brain of Neotermes castaneus (Fig. 1D) (Kalotermiti- trajectories. One of them was very loose and consisted of dae) harboured two triads of Crz-ir cells in the fronto-lateral a network of fibres that occupied a considerable part of the protocerebrum (Fig. 1Da) and 4 additional neurones in the fronto-lateral protocerebrum. Two deutocerebral cells in pars intercerebralis (PI) (Fig. 1Db). Fibres of the lateral cells both species, and 6 additional perikarya in the most lateral conjoined in two strongly stained trajectories that fused in brain region of C. formosanus (Fig. 2Bc) contributed to the medial part of protocerebrum into a single weakly stained this network. Another trajectory began in the lateral brain bundle connecting brain hemispheres via the protocerebral as a single fibre that split near the central body into loose fi- bridge. The bundle included projections of the PI cells bres with varicosities; the fibres came closer together in the (Fig. 1D). The lateral protocerebrum was characterised by a fi- brain midline where they linked the hemispheres. A third tra- bre network that expanded from the frontal to the medial brain jectory began also as a single fibre originating in the lateral region. Three fibres from this network ran through the deuto- cells. It ran frontally but looped back and continued ventrally cerebrum, ramified in the tritocerebrum, and apparently contin- to the deuto- and tritocerebrum. Processes of the PI cells ued to a network in the FG. No connection to perikarya could joined the loop in C. formosanus but no such connection be found in the strongly stained fibres running to the CC where was detected in R. flavipes. Also in C. formosanus, fibres de- they dispersed and terminated. A single Crz-ir fibre in the cir- scending from the loops to the ventral protocerebrum sent cumesophageal connective linked the tritocerebrum with the connections to the opposite hemisphere. In the tritocerebrum
Fig. 1. Diagrams illustrating the topology of Crz-ir perikarya and fibres in the brain-SOG complex of (A) Mastotermes darwiniensis, (B) Zootermopsis angusti- collis, (C) Hodotermes mossambicus, (D) Neotermes castaneus, and (E) Prorhinotermes simplex. Two cell clusters (arrows), each with 3 Crz-ir somata, in fronto- lateral protocerebrum of M. darwiniensis (Aa, Ab). Homologous clusters in Z. angusticollis (Ba, Bb); one of such clusters (Ca) and axon arborisation in the CC (Cb) of H. mossambicus. Crz-ir somata in the protocerebral clusters (arrows) (Da), pars intercerebralis (Db), and SOG (Dc) of N. castaneus. Frontolateral groups of perikarya in the whole mount (Ea) and a section (Eb) of P. simplex brain. Corpora allata (CA), corpora cardiaca (CC), deutocerebrum (DE), frontal ganglion (FG), pars intercerebralis (PI), protocerebrum (PR), optic lobe (OL), suboesophageal ganglion (SOG), tritocerebrum (TR). Scale bar 100 mm in Ca, Cb, Db, Dc, Eb, and 500 mm in Aa, Ab, Ba, Bb, Da, and Ea. Author's personal copy
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R. Za´vodska´ et al. / Arthropod Structure & Development 37 (2008) 273e286 279 of both species, the fibre trajectory descending from the loop, 3.2. PDF-immunoreactivity (PDF-ir) the processes of two deutocerebral perikarya, and axons from the fronto-lateral network, formed a spindle-like arrangement No PDF-ir was detected in the brains of workers of M. dar- of loose fibres with varicosities. The fibres continued to winiensis and only 2 small cells were located in the proximal a branching in the FG, and two of them possibly extended fronto-ventral (Pfv) part of the optic lobe (OL) in some ani- to the SOG. It could not be determined whether these 2 mals (Fig. 3Aa). Weakly stained projections of these cells axon bundles descended from the brain or ascended from formed a tiny bilateral network in the frontal protocerebrum the SOG that harboured two pairs of Crz-ir neurones located (Fig. 3A). dorsally in the mandibular neuromere in both species and 6 Z. angusticollis was the only examined termite species that additional perikarya in the ventral part of the labial neuro- contained PDF-ir perikarya in the protocerebrum. Each hemi- mere in C. formosanus. Crz-ir fibres branching in the SOG sphere contained 8 somata in the PI, a couple of unequally obviously connected the brain with the ventral nerve cord. large fronto-lateral cells, and a single big cell in extreme It is not excluded that Crz-ir fibres detected in the cardiacal fronto-lateral position close to the OL (Fig. 3B,Ba). Projec- nerves included projections from the SOG somata. The Crz-ir tions from these three cell groups formed a tract that passed staining of the cardiacal nerves was very distinct in R. fla- above the mushroom body, formed a lateral and a medial fibre vipes, in which some fibres ramified in the frontal CC region network in the frontal protocerebrum, and connected the brain and others projected to the caudal part of the CA. C. formo- hemispheres through the bridge. Some fibres arborized in the sanus contained slightly stained fibres in the CC and no mushroom body. A cluster of 4 PDF-ir perikarya in the Pfv staining in CA. area of OL sent fibres in a fan-like arrangement over the fron- Macrotermes jeanneli (Fig. 2C) and Odontotermes feae tal surface of the medulla to the base of the lamina. Other fi- (Fig. 2D) (both from Termitidae: Macrotermitinae) possessed bres ran in opposite direction and merged into a distinct 3 perikarya in a lateral (Fig. 2Ca,Db,Dc) and 3 in a medial connective to the tritocerebrum, where the fibres dispersed (Fig. 2Cb) cluster in the frontal protocerebrum. Nasutitermes in a spindle-like formation. Some fibres continued to a fine costalis (Fig. 2E) (Termitidae: Nasutermitinae) also contained ramification in FG and others to a small network located in two protocerebral clusters of Crz-ir cells (Fig. 2Ea), of which the dorso-frontal SOG close to a pair of PDF-ir somata in a group of 3 cells was in a fronto-lateral position and a group the mandibular neuromere. Fine fibres of unknown origin of 4 cells in PI. An additional group of 3 somata in the PI ran through the CC and arborized slightly in the proximal region (Fig. 2Da) and a pair of cells in the lateral brain part of the CA. (Fig. 2Dd) was found only in O. feae. Major Crz-ir fibre tra- H. mossambicus (Fig. 3C) harboured a ventral subgroup of jectories were alike in all three termitid species. One trajec- 2 and a somewhat more dorsally located subgroup of 4 PDF-ir tory dispersed into a network of fibres in the protocerebral cells in the Pfv region of OL. Few processes arising from the neuropile and extended with a few fibres to the opposite latter subgroup ran to the edge of medulla but not onto its sur- hemisphere. The network was very large and rich in varicos- face (Fig. 3Ca). A thin fibre extending from this subgroup in ities in N. costalis. Another trajectory of compound fibres the opposite direction proceeded dorsally, turned to the brain connected the hemispheres through the protocerebral bridge centre and passed through protocerebral bridge to the opposite and the dorsal part of central body. A third trajectory, which hemisphere (Fig. 3C). Very fine side branches of this trajectory began as a loop around the corpora pedunculata in M. jean- formed a narrow-range network in the frontal protocerebrum. neli and as an extension of the fronto-lateral fibre network in A second fibre trajectory originated in both Pfv subgroups, the two other species, linked the hemispheres in the ventral ramified in the posterio-ventral protocerebrum, and trans- protocerebrum. Finally, 2e3 fibres ran from the protocere- versed the ventral protocerebrum to the opposite OL. PDF-ir brum to the deutocerebrum, arborized in a spindle-like for- fibres in nervi corporis cardiaci and in the periphery of the mation in the tritocerebrum, and ramified in the FG. In O. CC were probably derived from this trajectory that also sent feae and N. costalis some fibres passed through the suboeso- a branch to a network in the tritocerebrum. Most fibres contin- phageal connectives, dispersed in the dorso-lateral SOG, and ued to a strongly stained ramification in the FG (Fig. 3Cb). A continued to the ventral nerve cord. In N. costalis they separate fibre passed through the circumoesophageal connec- seemed to connect to the 2 pairs of Crz-ir perikarya in the tive to the SOG where it entered in a ventrally located fibre labial neuromere (Fig. 2Eb). In all three species, the commis- network extending from a soma in the labial neuromere sure at the base of the protocerebrum sent branches to an ex- (Fig. 3Cc). tensive Crz-ir ramification in the CC (Fig. 2Cc). No staining Our investigations on N. castaneus confirmed the data pub- was found in the CA. lished by Sehadova´ et al. (2003). N. castaneus was the only
Fig. 2. The topology of Crz-ir neurones in the brain-SOG complex of (A) Reticulitermes flavipes, (B) Coptotermes formosanus, (C) Macrotermes jeanneli, (D) Odontotermes feae, and (E) Nasutitermes costalis. Crz-ir cells in the lateral protocerebrum (Aa) and SOG (Ab) of R. flavipes. Cry-or cells in frontolateral proto- cerebrum (Ba, arrow), pars intercerebralis (Bb), and most lateral protocerebrum (Bc) of C. formosanus. Perikarya in frontolateral (Ca) and frontomedial (Cb) protocerebrum, and fibres in CC (Cc) of M. jeanneli. Somata in pars intercerebralis (Da), frontolateral protocerebrum (Db, Dc), and lateral protocerebrum (Dd) of O. feae. Perikarya in pars intercerebralis and frontolateral protocerebrum (Ea) and in SOG (Eb) of N. costalis. Corpora cardiaca (CC), pars intercerebralis (PI), protocerebrum (PR), suboesophageal ganglion (SOG). Scale bar 500 mm in Ba and 100 mm in all other pictures. Author's personal copy
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R. Za´vodska´ et al. / Arthropod Structure & Development 37 (2008) 273e286 281 termite in our study with three clusters of PDF-ir neurones in tritocerebrum, FG, and CC, and a pair of perikarya in the la- the OL: a Pfv cluster of 8 cells and a distal posterio-dorsal and bial neuromere with processes extending to the brain distal posterio-ventral clusters each of 10 cells (Fig. 3Da,Db). (Fig. 4CeE). M. jeanelli (Fig. 4C) contained a Pfv cluster of In addition, 2 smaller cells occurred in the ventro-lateral pro- 4 small PDF-ir perikarya (Fig. 4Ca) with centripetal neurites tocerebrum, and 2 pairs of large dorso-lateral cells in the man- that formed an indistinct network in the frontal protocerebrum dibular, and a medial group of 5 cells in the maxillary and a hardly recognizable link to the opposite hemisphere. neuromere of SOG. The positions of perikarya and the course Delicate fibres arborizing in the tritocerebrum had no visible of PDF-ir fibres depicted in Fig. 3D were consistent with the connection to the protocerebrum but could be traced to the published account. FG (Fig. 4Cb) and through the circumoesophageal connectives All three species of Rhinotermitidae possessed 1e2 pairs of to the bilateral SOG networks and to perikarya in the labial PDF-ir somata in the labial neuromere of the SOG and a rich neuromere. Fine fibres were detected in the cardiacal nerves PDF-ir innervation in the tritocerebrum, FG and SOG, but dif- and the CC (Fig. 4Cc). O. feae (Fig. 4D) possessed 4e5 Pvf fered from one another in the PDF-ir distribution in the proto- cells located more ventrally and proximally than in the other cerebrum and optic lobes (Figs. 3E and 4A,B). The Pfv region species (Fig. 4Da). A short fibre supplied a fan of processes of optic lobes contained 3 PDF-ir neurones in P. simplex dispersed over the medulla surface towards the lamina. An- (Fig. 3Ea), none in M. jeanneli, and 4 in C. formosanus. other projection of these cells ran in the opposite direction When present, some processes of these neurones formed a sim- and branched into a network spreading in the frontal proto- ple fan on the frontal medulla face (Figs. 3E and 4B), while cerebrum over both hemispheres (Fig. 4D). A third projection others extended to the protocerebrum. Two tracts ran towards starting from the Pvf group ran ventrally, formed a loose bun- the brain centre in P. simplex. One started in a dorsal direction, dle of varicose fibres in the ventral protocerebrum, continued formed a network in the dorso-lateral protocerebrum, and con- to a dense plexus in the tritocerebrum, and eventually formed tinued above the protocerebral neuropile to the brain midline a network in the FG (Fig. 4Db,Dd). The innervation of trito- without making a discernible connection to the opposite hemi- cerebrum and FG, as well as the PDF-ir fibres in the cardiacal sphere. The second trajectory dispersed in the fronto-ventral nerves and in the proximal part of CC, are connected to pro- protocerebrum without any links to the opposite hemisphere cesses traversing the circumoesophageal connectives and ap- or to a network of strongly stained fibres in the tritocerebrum. parently originating in perikarya in the labial neuromere The tritocerebral fibres possibly originated in SOG that had (Fig. 4Dc). N. costalis (Fig. 4E) was characterised by two sub- a clear connection via the PDF-ir fibres in the FG. The fibres groups of Pfv cells in the OL. Two cells were located ventrally running through SOG formed slight ramifications and con- to a group of 4 cells (Fig. 4Ea) that formed a simple fan of nected to a pair of perikarya in the labial neuromere (Fig. 3Eb). weekly stained fibres on the frontal medulla face. Centripetal P. simplex had no PDF-ir in the CC. fibres from both subgroups adjoined in a trajectory that split In C. formosanus, several fibres extended from 4 perikarya into a dorsal tract passing the frontal, and a ventral track pass- in the Pfv cluster (Fig. 4Bb) over the fronto-dorsal protocere- ing the posterior protocerebrum. The stronger frontal branch brum, while others formed a high-density network in the linked the brain hemispheres via the protocerebral bridge fronto-medial protocerebrum and continued to a strongly and supplied fibres to a large network extending over most stained plexus in the tritocerebrum (Fig. 4Bc). The plexus of the frontal protocerebrum. A fibre leaving this network sent processes through the frontal connectives to a fibre rami- and a posterio-ventral branch of the initial track travelled fication in the FG and through the circumoesophageal connec- around the deutocerebrum, arborized in the tritocerebrum, tives to loose arborizations and a pair of large perikarya in the and dispersed in a FG plexus. A pair of perikarya in the labial SOG (Fig. 4Ba). Several PDF-ir axons from unidentified peri- neuromere (Fig. 4Eb) sent neurites to a tiny arborization in the karya terminated in the CC. SOG and further on to the circumoesophageal connectives and R. flavipes differed from both other species by the absence possibly to the tritocerebrum, FG, and proximal CC. of PDF-ir in the protocerebrum and optic lobes (Fig. 4A) and the presence of two (rather than one) pairs of PDF-ir neurones 4. Discussion in the labial neuromere of the SOG (Fig. 4Aa). The processes of these perikarya were traced to the circumoesophageal con- 4.1. The distribution of Crz-ir in the cephalic ganglia nectives and probably provided PDF-ir innervations to the tri- of termites tocerebrum and FG. The CC were devoid of PDF-ir. All examined Termitidae possessed bilaterally connected Crz has not been isolated from the termites chemically and Pfv clusters of 4e6 PDF-ir neurones, fibre networks in the no termite gene encoding Crz has been described. However,
Fig. 3. The topology of PDH-ir neurones in the brain-SOG complex of (A) Mastotermes darwiniensis, (B) Zootermopsis angusticollis, (C) Hodotermes mossam- bicus, (D) Neotermes castaneus, and (E) Prorhinotermes simplex. Two cells in OL of M. darwiniensis (Aa). PDH-ir perikarya in the brain of Z. angusticollis (Ba): arrows indicate from right to left cells in pars intercerebralis, a couple of frontolateral cells, a single big cell in frontolateral position, and a cluster of 4 cells in the Pvf area of OL. Somata in Pfv region (Ca), fibres in FG (Cb), and a soma in SOG (Cc) of H. mossambicus. Three clusters (arrows) of neurones in OL (Da) and a close up of the Pfv cluster (Db, arrow) in N. castaneus. Pfv cluster of three cells in OL (Ea, arrow) and a single PDH-ir soma in SOG (Eb) of P. simplex. Frontal ganglion (FG), protocerebrum (PR), medulla (me), optic lobe (OL), suboesophageal ganglion (SOG), tritocerebrum (TR). Scale bar 100 mm in Aa, Ca, Cb, Cc, Db, Eb; 500 mm in Ba, Da, and Ea. Author's personal copy
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Table 2 Pairs of Crz-ir perikarya and the location of Crz-ir fibres in the Br-SOG complex Species Perikarya Network of fibres F-L Pr De PI SOG F-LPr PB CB De Tr FG CC CA SOG Mastotermes darwiniensis 3 þ 3000 þ þþ þ Zootermopsis angusticollis 3 þ 3000 þ þ þ Hodotermes mosambicus 3 þ 3000 þ þþþþþþþ Neotermes castaneus 3 þ 3042þ 2 þ 2/3 þ þ þþþþ þ Prorhinotermes simplex 3 þ 3 þ 20 0 0 þ þþ þþþþ Reticulitermes flavipes 3 þ 3242 þ þþþþþþþþ Coptotermes formosanus 3 þ 3 þ 62 4 2þ 1 þ 2/3 þ þþþþþþ þ Macrotermes jeanneli 3 þ 3000 þ þþþþþþ Odontotermes feae 3 þ 3 þ 20 3 0 þ þþþþþþ þ Nasutitermes costalis 3042 þ þþþþþþ þ CA, corpora allata. CB, central body; CC, corpora cardiaca; De, deutocerebrum; FG, frontal ganglion; F-L Pr, fronto-lateral protocerebrum; PB, protocerebral bridge; PI, pars intercerebralis; SOG, suboesophageal ganglion; Tr, tritocerebrum. structural conservation of Crz in various insects (Veenstra, neurone at the base of optic lobe. No Crz-ir cells were found 1989, 1991, 1994; Predel et al., 1999; Tawfik et al., 1999; in this location in termites. Hua et al., 2000; Hansen et al., 2001; Roller et al., 2006; Ver- The occurrence of Crz-ir perikarya in the PI and SOG is leyen et al., 2006) suggests strongly that it is also present in rather exceptional. Weekly stained Crz-ir cells in the PI Isoptera. Crz-ir in the cephalic ganglia of termites is confined were found only in crickets (Sehadova´ et al., 2007) and the to specific neurones. Three to 14 Crz-ir cells occur in the caterpillars of Bombyx mori (Qi-Miao et al., 2003). Crz-ir so- fronto-lateral protocerebrum of all 10 species and additional mata in the SOG were described in the American cockroach 3e4 cells in the PI of 5 species (Table 2). Two (only one in (Veenstra and Davis, 1993), two cricket species (Sehadova´ N. costalis) triads of somata in the fronto-lateral protocere- et al., 2007), the silverfish Ctenolepisma lineata, and the bug brum and three principal courses of their fibres are highly con- Pyrrhocoris apterus (Roller et al., 2003). On the other hand, served (Figs. 1 and 2). One fibre tract links homologous cells the SOG of nearly all insects contains Crz-ir fibres linking in the opposite hemisphere, another forms a protocerebral net- the brain with the ventral nerve cord. These fibres are missing work, and a third one projects into the base of the protocere- in some crickets (Sehadova´ et al., 2007) and in four termite brum, CC and, with separate branches, often also to the species (Table 2). deuto- and tritocerebrum and FG. N. castaneus, R. flavipes and C. formosanus, and all Termitidae species are distin- 4.2. The distribution of PDF-ir in the cephalic ganglia guished by rich fibre ramifications in the tritocerebrum. The of termites occurrence of Crz-ir perikarya in the SOG was limited to 4 species but Crz-ir fibres were detected in this ganglion in 6 Four out of 18 amino acid residues vary in the PDF homo- species (Table 2). logues that have been identified in diverse insects (reviewed by The presence of Crz-ir perikarya in the fronto-lateral proto- Hamasaka et al., 2005). The homologues usually differ only cerebrum with projections to the ipsilateral corpora cardiaca by two replacements, mostly with amino acids of similar prop- was described in diverse insects (Roller et al., 2003; Hama- erties. Termites are likely to contain [Leu8, Leu15]-PDF that naka et al., 2004; Choi et al., 2005; and references therein) was found in cockroaches (Mohrherr et al., 1991; Hamasaka and probably represents an ancient pattern. The arrangement et al., 2005) and a cricket (Singaravel et al., 2003). The poly- of protocerebral Crz-ir somata in two triplets seems to be clonal antiserum employed in our study was shown to react unique to the termites. It somewhat resembles the silverfish with cells that contain different PDF homologues (Sehadova´ Ctenolepisma lineata, which contains three groups Crz-ir peri- et al., 2003; Honda et al., 2006). karya in the lateral protocerebrum (Roller et al., 2003) similar The proximal fronto-ventral (Pfv) region of the OL is a typ- to N. castaneus, P. simplex, and O. feae. Closer relatives of ter- ical location of the PDF-ir neurons in insects, including ter- mites, such as the cockroach Leucophaea maderae (Petri et al., mites; complete absence of PDF-ir perikarya in the OL of R. 1995), a locust and a grasshopper (Roller et al., 2003), and two flavipes is unique (Table 3). The Pfv group in termites typi- cricket species (Sehadova´ et al., 2007) harbour a Crz-ir cally consists of 2e6 somata but in N. castaneus it includes
Fig. 4. The topology of PDH-ir neurones in the brain-SOG complex of (A) Reticulitermes flavipes, (B) Coptotermes formosanus, (C) Macrotermes jeanneli, (D) Odontotermes feae, and (E) Nasutitermes costalis. One (arrow) of two somata in SOG of R. flavipes (Aa). PDH-ir cells in SOG (Ba), Pfv cluster of four cells in OL (Bb), and fibres in the tritocerebrum (Bc) of C. formosanus. Four Pfv cells in OL (Ca), fibres in frontal ganglion (Cb), and in the corpora cardiaca (Cc) of M. jeanneli. Pfv cells in OL (Da), fibre arborisation in FG (Db), perikarya in SOG (Dc), and fibres ramifying in the tritocerebrum (Dd) of O. feae. Two subgroups of Pfv cells in OL (Ea, arrows) and perikarya in the SOG (Eb arrows) of N. costalis. Corpora cardiaca (CC), frontal ganglion (FG), medulla (me), optic lobe (OL), protocerebrum (PR), suboesophageal ganglion (SOG), tritocerebrum (TR). Scale bar 100 mm in Aa to Dd and 500 mm in Ea and Eb. Author's personal copy
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Table 3 Pairs of PDF-ir perikarya and the occurrence of PDF-ir fibres in different regions of the Br-SOG complex Species Perikarya Networks of fibres Pr OL SOG Pr PB CB Me La Tr FG CC CA SOG Mastotermes darwiniensis 02 0þþ Zootermopsis angusticollis 8 þ 2 þ 14 1 þþ þþþþþþþ Hodotermes mosambicus 06 1þþ þ þþþ þ Neotermes castaneus 28þ 10 þ 10 2 þ 2/3 þþ þþþþþ þ Prorhinotermes simplex 03 1þþ þþþþ þ Reticulitermes flavipes 00 2 þþ þ Coptotermes formosanus 04 1þ þþþþþ þ Macrotermes jeanneli 04 1þþ þþþ þ Odontotermes feae 05 1þþ þþþþþ þ Nasutitermes costalis 06 1þþ þþþþþ þ Pr, protocerebrum; SOG, suboesophageal ganglion; OL, optic lobe; PB, protocerebral bridge; CB, central body; Me, medulla; La, lamina; Tr, tritocerebrum; FG, frontal ganglion; CC, corpora cardiaca; CA, corpora allata.
10 cells (Figs. 3 and 4). This species possesses similar groups insects. This is consistent with the view that Isoptera represent of PDF-ir cells also in a distal posterio-dorsal (Dpd) and a pos- a monophyletic clade derived from cockroaches (Lo et al., terio-ventral (Dpv) region of the OL, in accordance with the 2000). Mastotermitidae, with a sole extant species M. darwin- report by Sehadova´ et al. (2003). The Pfv, Dpd and Dpv clus- iensis, are the most basal family. Termopsidae and Hodotermiti- ters of PDF-ir perikarya were found in various species of the dae may be sister families that evolved in parallel to cohort Polyneoptera (Homberg et al., 1991; Na¨ssel et al., Kalotermitidae; all these families are basal to Rhinotermitidae 1991; Stengl and Homberg, 1994); only a stonefly (Plecoptera) that may be polyphyletic. Termitidae are regarded as a monophy- and a praying mantis (Mantodea) lacked the Dpd and Dpv letic and most apical termite family, with Nasutermitinae at the clusters, similar to all examined species of the cohorts Zygen- very top (Kambhampati and Eggleton, 2000; Eggleton, 2001). toma, Odonata, Paraneoptera, and Holometabola (Sehadova´ Comparison of species from different termite families and et al., 2003; Za´vodska´ et al., 2003). Pfv neurons of diverse in- subfamilies indicates certain trends in the evolution of the sects typically send centripetal fibres into the protocerebrum Crz-ir and PDF-ir systems. Since Mastotermitidae, Termopsi- and a fan of centrifugal fibres embracing the medulla surface. dae, and Hodotermitidae contain just 6 Crz-ir perikarya in the This arrangement is conserved in most termites but the course protocerebrum, the presence of additional Crz-ir neurons in of fibres in the protocerebrum is diversified (Figs. 3 and 4). the brain and SOG in the more advanced families is apparently The protocerebrum of most termites is devoid of PDF-ir peri- an apomorphic character (Figs. 1 and 2). The processes of karya (Table 3), similar to most other Polyneoptera (Homberg these Crz-ir perikarya in the brain and SOG adjoin the estab- et al., 1991; Sehadova´ et al., 2003). The abundance of PDF-ir lished fibre tracts. Another feature shared by Kalotermitidae, neurons in the protocerebrum of Z. angusticollis is exceptional Rhinotermitidae (except P. simplex), and Termitidae is the tri- for the entire cohort. tocerebral arborization of the Crz-ir fibres. The PDF-ir system A characteristic feature of termites is the occurrence of of termites is distinguished from that of most other Polyneo- PDF-ir somata in the SOG and their association with rich fi- ptera by a reduced number of PDF-ir neurons in the OL bre arborization in the deuto- and tritocerebrum and the FG and, with the exception of Mastotermitidae, a rich PDF-ir (Table 3). Absence of this PDF-ir system in M. darwiniensis innervation of the tritocerebrum and FG and the presence of could be related to the old age of the analyzed workers and PDF-ir perikarya in the SOG. Neither the cause nor the conse- must be verified. Outside termites, PDF-ir somata in the quences of these evolutionary changes are obvious. The ter- SOG were found in the walking stick and in cockroaches of mites live in darkness and the pseudergates/workers of most the Blaberidae family (Sehadova´ et al., 2003). The fibre ram- species are blind. Reduced eyes are retained in the pseuder- ifications in the deuto- and tritocerebrum and the dispersal of gates (and soldiers) only in Kalotermitidae and Termopsidae. fibres in the FG seem to be specific for termites. In seven ter- These termites build single-site nests, Kalotermitidae in dry mite species we found PDF-ir axons passing through nervi wood, such as dead tree branches, and Termopsidae in damp corporis cardiaci to the CC (Table 3), indicative of PDF rotting wood. It may be significant that the pseudergates of release into the haemolyph. Among Polyneoptera, a similar N. castaneus from Kalotermitidae contain three clusters of projection of the PDF-ir fibres into the CC was described PDF-ir perikarya at the base of the optic lobe and that Z. an- only in a walking stick (Sehadova´ et al., 2003). gusticollis representing Termopsidae possesses an elaborate and unique PDF-ir system in the protocerebrum. 4.3. Correlation between Crz-ir and PDF-ir distribution and termite phylogeny Acknowledgements
The distributions of both Crz-ir and PDF-ir in the cephalic We cordially acknowledge the gift of termites by Prof. emer- ganglia of termites exhibit several features absent in other itus Reinhard Leuthold (Ittigen, Switzerland) and Mr. Marc Author's personal copy
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Rosset (Zoological Garden, Bern, Switzerland), by Dr. Jan Krˇe- Kambhampati, S., Eggleton, P., 2000. Phylogenetics and taxonomy. In: cek (Ft. Lauderdale REC University of Florida, USA) and by Abe, T., Bignell, D.E., Higashi, M. (Eds.), Termites: Evolution, Social- Dr. Horst Hertel (Bundesanstalt fur Materialprufung, Berlin, ity, Symbioses, Ecology. Kluwer Academic Publishers, Dordrecht, ¨ ¨ pp. 1e23. Germany). Our thanks are further due to Dr. Yupa Hanboonsong Kim, Y., Spalovska´, J., Valachova´, I., Cho, K.H., Zˇ itnˇanova´, I., Park, Y., (Khon Kaen University, Thailand) who kindly arranged and Adams, M.E., Zˇitnˇan, D., 2004. Corazonin receptor signaling in ecdysis oversaw the field collection of termites in Thailand. Dr. S. Ta- initiation. Proceedings of the National Academy of Sciences of the USA naka (Institute of Agrobiological Sciences, Tsukuba, Japan) 101, 6704e6709. generously provided the antiserum to Crz and Dr. H. Dircksen Lo, N., Tokuda, G., Watanabe, H., Rose, H., Slaytor, M., Maekawa, K., Bandi, C., Noda, H., 2000. Evidence from multiple gene sequences (Bonn University, Germany) the antiserum to PDH. This study indicates that termites evolved from wood-feeding cockroaches. Current was performed in frame of projects MSMT 6007665801 (R. Za´- Biology 10, 801e804. vodska´) and Z4 055 0506 (I. Hrdy´), and was supported by grants Lupien, M., Marshall, S., Leser, W., Pollack, G.S., Honegger, H.-W., 2003. MSMT LC07032 (I. Sauman) and NSC 92-2313-B-002-069, Antibodies against the PER protein of Drosophila label neurones in the op- NSC 93-2313-B-002-045, NSC 2313-B-002-015 (H.-J. Lee). tic lobe, central brain, and thoracic ganglia of the crickets Teleogryllus commodus and Teleogryllus oceanicus. Cell and Tissue Research 312, 377e391. Mohrherr, C.J., Rao, K.R., Riehm, J.P., 1991. Characterization of a pigment- References dispersing factor from the American cockroach. Society Neuroscience Ab- stracts 17, 276. Billimoria, C.P., Li, L., Marder, E., 2005. Profiling of neuropeptides released Na¨ssel, D.R., Shiga, S., Wikstrand, E.M., Rao, K.R., 1991. Pigment-dispersing at the stomatogastric ganglion of the crab Cancer borealis with mass spec- hormone-immunoreactive neurons and their relation to serotoenergic neu- trometry. Journal of Neurochemistry 95, 191e199. rons in the blowfly and cockroach visual system. Cell and Tissue Research e Choi, Z.L., Lee, G., Hall, J.C., Park, J.H., 2005. Comparative analysis of cor- 266, 511 523. azonin-encoding genes (Crz’s) in Drosophila species and functional in- Petri, B., Stengl, M., 1997. Pigment-dispersing hormone phase-shifts the sights into Crz-expressing neurons. Journal of Comparative Neurology circadian pacemaker of the cockroach Leucophaea maderae. Journal of e 482, 372e385. Neuroscience 17, 4087 4093. Dircksen, H., Zahnow, C.A., Gaus, G., Keller, R., Rao, K.R., Riem, J.P., 1987. Petri, B., Stengl, M., Wu¨rden, S., Homberg, U., 1995. Immunocytochemical The ultrastructure of nerve endings containing pigment-dispersing hor- characterization of the accessory medulla in Leucophaea maderae. Cell e mone (PDH) in crustacean glands: identification by an antiserum against and Tissue Research 282, 3 19. a synthetic PDH. Cell and Tissue Research 250, 377e387. Predel, R., Agricola, H., Linde, D., Wollweber, L., Veenstra, J.A., Penzlin, H., Eggleton, P., 2001. Termites and trees: a review of recent advances in termite 1994. The insect neuropeptide corazonin: physiological and immunocyto- e phylogenetics. Insectes Sociaux 48, 187e193. chemical studies in Blattaria. Zoology 98, 35 49. Hanus, R., Sˇobotnı´k, J., 2004. The ontogeny of termites (Isoptera) in the con- Predel, R., Kellner, R., Ga¨de, G., 1999. Myotropic neuropeptides from the ret- text of ecological strategies. Biologicke´ listy 69, 29e57 (in Czech). rocerebral complex of the stick insect, Carausius morosus (Phasmatodea: e Hamanaka, Y., Numatu, H., Shiga, S., 2004. Morphology and electrophysio- Lonchodidae). European Journal of Entomology 96, 275 278. Qi-Miao, S., Tanaka, S., Takeda, M., 2003. Immunocytochemical localiza- logical properties of neurons projecting to the retrocerebral complex in 7 7 the blow fly, Protophormia terraenovae. Cell and Tissue Research 318, tion of clock proteins (DBT and PER), and [His ]- and [Arg ]-corazo- 403e418. nins in the cerebral ganglia of Bombyx mori: are corazonins Hamasaka, Y., Mohrherr, C.J., Predel, R., Wegener, C., 2005. Chronobiologi- downstream regulators of circadian clocks? European Journal of Ento- e cal analysis and mass spectrometric characterization of pigment-dispersing mology 100, 283 286. factor in the cockroach Leucophaea maderae. Journal of Insect Science 5- Rao, K.R., Riehm, J.P., Zahnow, C.A., Kleinholz, L.H., Tarr, G.E., Johnson, L., 43, 1e13. Norton, S., Landau, M., Semmes, O.J., Sattelberg, R.M., Jorenby, W.H., Hansen, I.A., Sehnal, F., Meyer, S., Scheller, K., 2001. Corazonin gene expres- Hintz, M.F., 1985. Characterization of a pigment-dispersing hormone in sion in the wax moth Galleria mellonella. Insect Molecular Biology 10, eyestalks of the fiddler crab Uca pugilator. Proceedings of the National e 341e346. Academy of Sciences of the USA 82, 5319 5322. Helfrich-Fo¨rster, C., Homberg, U., 1993. Pigment-dispersing-hormone-immu- Renn, S.C.P., Park, J.H., Rosbash, M., Hall, J.C., Taghert, P.H., 1999. A pdf noreactive neurons in the nervous system of wild-type Drosophila mela- neuropeptide gene mutation and ablation of PDF neurons both cause se- nogaster and of several mutants with altered circadian rhythmicity. vere abnormalities of circadian behavioral rhythms in Drosophila. Cell e Journal of Comparative Neurology 337, 177e190. 99, 791 802. Helfrich-Fo¨rster, C., Winter, C., Holbauer, A., Hall, J.C., Stanewsky, R., 2000. Roisin, Y., 2000. Diversity and evolution of caste patterns. In: Abe, T., The circadian clock of fruit flies is blind after elimination of all known Bignell, D.E., Higashi, M. (Eds.), Termites: Evolution, Sociality, Symbio- e photoreceptors. Neuron 30, 249e261. ses, Ecology. Kluwer Academic Publishers, Dordrecht, pp. 95 119. Homberg, U., Wu¨rden, S., Dircksen, H., Rao, K.R., 1991. Comparative Roller, L., Tanaka, Y., Tanaka, S., 2003. Corazonin and corazonin-like sub- anatomy of pigment-dispersing hormone-immunoreactive neurons in the stances in the central nervous system of the Pterygote and Apterygote in- e brain of orthopteroid insects. Cell and Tissue Research 266, 343e357. sects. Cell and Tissue Research 312, 393 406. Honda, T., Matsushima, A., Sumida, K., Chuman, Y., Sakaguchi, K., Roller, L., Tanaka, S., Kimura, K., Satake, H., Tanaka, Y., 2006. Molecular Onoue, H., Meinertzhagen, I.A., Shimohigashi, Y., Shimohigashi, M., cloning of [Thr(4), His(7)]-corazonin (Apime-corazonin) and its distribu- 2006. Structural isoforms of the circadian neuropeptide PDF expressed tion in the central nervous system of the honey bee Apis mellifera (Hyme- e in the optic lobes of the cricket Gryllus bimaculatus: immunocytochemical noptera: Apidae). Applied Entomology and Zoology 41, 331 338. evidence from specific monoclonal antibodies. Journal of Comparative Sehadova´, H., Sauman, I., Sehnal, F., 2003. Immunocytochemical distribution Neurology 499, 404e421. of the pigment-dispersing hormone in the cephalic ganglia of polyneo- e Hua, Y.-J., Ishibashi, J., Saito, H., Tawfik, A.I., Sakakibara, M., Tanaka, Y., pteran insects. Cell and Tissue Research 312, 113 125. Derua, R., Waelkens, E., Baggerman, G., De, Loof, A., Schoofs, L., Sehadova´, H., Shao, Q.-M., Sehnal, F., Takeda, M., 2007. Neurohormones as Tanaka, S., 2000. Identification of [Arg7]-corazonin in the silkworm, putative circadian clock output signals in the central nervous system of two e Bombyx mori, and the cricket, Gryllus bimaculatus, as a factor inducing cricket species. Cell and Tissue Research 328, 239 255. dark color in an albino strain of the locust, Locusta migratoria. Journal Singaravel, M., Fujisawa, Y., Hisada, M., Saifullah, A.S.M., Tomioka, K., of Insect Physiology 46, 853e859. 2003. Phase shifts of the circadian locomotor rhythm induced by Author's personal copy
286 R. Za´vodska´ et al. / Arthropod Structure & Development 37 (2008) 273e286
pigment-dispersing factor in the cricket Gryllus bimaculatus. Zoological Veenstra, J.A., 1991. Presence of corazonin in three insect species, and isola- Science 20, 1347e1354. tion and identification of [His7] corazonin from Schistocerca americana. Sla´ma, K., 2004. The effect of corazonin on heartbeat reversal in pupae of the Peptides 12, 1285e1289. tobacco hornworm, Manduca sexta (Lepidoptera Sphingidae). European Veenstra, J.A., 1994. Isolation and structure of the Drosophila corazonin gene. Journal of Entomology 101, 513e521. Biochemical and Biophysical Research Communications 204, 292e296. Stengl, M., Homberg, U., 1994. Pigment-dispersing hormone-immunoreactive Veenstra, J.A., Davis, N.T., 1993. Localization of corazonin in the nervous sys- neurons in the cockroach Leucophaea maderae share properties with circa- tem of the cockroach Periplaneta americana. Cell and Tissue Research dian pacemaker neurons. Journal of Comparative Physiology 175, 203e213. 274, 57e64. Taghert, P.H., Shafer, O.T., 2006. Mechanisms of clock output in the Drosophila Verleyen, P., Baggerman, G., Mertens, I., Vandersmissen, T., Huybrechts, J., circadian pacemaker system. Journal of Biological Rhythms 21, 445e457. Van Lommel, A., De Loof, A., Schoofs, L., 2006. Cloning and character- Tawfik, A.I., Tanaka, S., De Loof, A., Schoofs, L., Baggerman, G., ization of a third isoform of corazonin in the honey bee Apis mellifera. Waelkens, E., Derua, R., Milner, Y., Yerushalmi, Y., Pener, M.P., 1999. Peptides 27, 493e499. Identification of the gregarization-associated dark-pigmentotropin in lo- Watson, C.M., Sauman, I., Berry, S.J., 1993. Actin is a major structural and custs through an albino mutant. Proceedings of the National Academy of functional element of the egg cortex of giant silkmoth during oogenesis. Sciences of the USA 96, 7083e7087. Developmental Biology 155, 315e323. Thorne, B.L., Grimaldi, D.A., Krishna, K., 2000. Early fossil history of the termites. Wise, S., Davis, N.T., Tyndale, E., Noveral, J., Folwell, M.G., Bedian, V., In: Abe, T., Bignell, D.E., Higashi, M. (Eds.), Termites: Evolution, Sociality, Emery, I.F., Siwicki, K.K., 2002. Neuroanatomical studies of period Symbiosis, Ecology. Kluwer Academic Publishers, Dordrecht, pp. 77e94. gene expression in the hawkmoth, Manduca sexta. Journal of Comparative Truman, J.W., 2005. Hormonal control of insect ecdysis: endocrine cascades Neurology 447, 366e380. for coordinating behavior with physiology. Vitamins and Hormonesd Za´vodska´, R., Sauman, I., Sehnal, F., 2003. Distribution of PER protein, pig- Advances in Research and Applications 73, 1e29. ment-dispersing hormone, prothoracicotropic hormone, and eclosion hor- Veenstra, J.A., 1989. Isolation and structure of corazonin, a cardioactive pep- mone in the cephalic nervous system of insects. Journal of Biological tide from the American cocktroach. FEBS Letters 250, 231e234. Rhythms 18, 106e122.