Invertebrate Biology 133(3): 261–273. © 2014, The American Microscopical Society, Inc. DOI: 10.1111/ivb.12061

Chaetal loss and replacement in Pseudopotamilla reniformis (, Annelida)

Glafira D. Kolbasova,1,a Alexander B. Tzetlin,2 and Elena K. Kupriyanova3

1 Department of Invertebrate Zoology, Moscow State University, Moscow 119991, Russia 2 Pertsov White Sea Biological Station, Moscow State University, Moscow 119991, Russia 3 The Australian Museum, Sydney, New South Wales 2010, Australia

Abstract. Chaetae are continually being replaced during the life of a , but the process of chaetal degeneration during replacement is still poorly known. Chaetal loss occurs either one at a time through special degenerative sites, or all chaetae in a chaetal sac are lost simultaneously in the absence of degenerative sites. Old chaetae can be either resorbed inside the coelom or shed. This study describes chaetal degradation, loss, and sub- sequent replacement during experimentally induced regeneration in the sabellid polychaete Pseudopotamilla reniformis. During post-traumatic regeneration in P. reniformis, abdominal- type chaetae fall from the chaetal sac simultaneously without degenerative sites, and are replaced by thoracic-type chaetae. Chaetal sac degradation occurs in three main stages: (1) formation of numerous lysosomes and small electron-transparent vesicles in follicle cell cytoplasm; (2) disintegration of follicular cell membranes; and (3) disintegration of follicular cell cytoplasm. Description of changes during parapodial transformation allows identifica- tion of signs of degradation that can serve as histological indicators of chaetal degeneration in a wide range of . Our study suggests that chaetal degeneration and loss in polychaetes can occur either with degenerative sites or without them, depending on the type of chaetal replacement. Additional key words: chaetal sac, chaetal degeneration, chaetogenesis, morphallaxis, uncini

Chaetae, epidermal extracellular structures that replaced by those of another, as shown for, e.g., lent their name to the now-defunct taxon Polychae- Lumbrineris fragilis (O.F. MULLER€ 1766) by Tzetlin ta, play an important role in life of these (1990), Scolelepis squamata (MULLER€ 1806) by worms (Woodin & Merz 1987). Chaetal morphology Hausen & Bartolomaeus (1998), and Dipolydora and functions are extremely variable: in different concharum (VERRILL 1880) by Radashevsky & species, chaetae are used for defense, crawling, Fauchald (2000). In large polychaetes that grow dra- swimming, burrowing, and body anchoring within matically in their lifetime, small juvenile chaetae are tubes (Woodin & Merz 1987; Fitzhugh 1991; Merz replaced by larger adult chaetae (Pilgrim 1977; & Woodin 2006). In spite of the functional impor- Bhaud 1988; Duchene & Bhaud 1988). In addition, tance of chaetae, many aspects of chaetal develop- worn-out and broken adult chaetae are replaced by ment are still unclear. new chaetae (Hausen 2005). Annelid chaetae are continually and routinely There are, however, two unusual types of chaetal replaced during the life of a worm. During settle- replacement. Probably the most remarkable of them ment and metamorphosis, larval chaetae are is epitoky, metamorphosis of an immature worm replaced by juvenile chaetae (Eckelbarger 1975, into a sexually mature reproductive form. Epitokous 1976, 1977; Amieva & Reed 1987; Eeckhaut et al. transformation results in atokous chaetae of benthic 2003; Gibson & Gibson 2004; Gibson & Smith 2004; worms being replaced by modified epitokous swim- Pernet & McArthur 2006; Smart & von Dassow ming chaetae (see, e.g., Clark 1961; Simpson 1962; 2009; Budaeva & Fauchald 2010). As juveniles Hofmann 1974; Daly 1975; Kuper & Westheide grow, chaetae of one morphological type may be 1998; Petersen 1999; Chatelain et al. 2008). Another unusual type of chaetal replacement takes place dur- aAuthor for correspondence. ing regeneration of and Serpulidae. The E-mail: [email protected] body of these worms is subdivided into thorax and 262 Kolbasova, Tzetlin, & Kupriyanova abdomen, and the thoracic chaetal arrangement dif- tion in this species can be easily induced artificially fers from the abdominal one. A part of an abdomen (Kolbasova et al. 2013). Such experimentally can regenerate into a complete worm; during regen- induced regeneration provides a convenient experi- eration, anterior abdominal parapodia are trans- mental system not only for studies of chaetal regen- formed into thoracic ones by losing their old eration (Berrill 1977, 1978; Murray 2010; Licciano abdominal-type chaetae and replacing them with et al. 2012), but also for examination of degenera- thoracic-type chaetae (Berrill 1931, 1977, 1978; tive sites. Berrill & Mees 1936a,b; Pernet 2001; Murray 2010; Licciano et al. 2012). Methods Each chaeta develops from an individual epider- mal follicle. Several follicles are usually organized in The adult body length of Pseudopotamilla renifor- one or several rows situated inside a common chae- mis is 50–70 mm. The thorax consists of 7–14 (typi- tal sac, an invagination of epithelial basal lamina cally 8–9) segments, and the abdomen consists of (Bartolomaeus 1995; Hausen 2005; Kieselbach & 80–120 chaetigers. Both thoracic and abdominal Hausen 2008). Usually one parapodium has two parapodia are illustrated in Fig. 1A–D. The first chaetal sacs, the notopodial sac and the neuropodial (collar) segment lacks neuropodia, while the subse- one (Mettam 1967; O’Clair & Cloney 1974). Accord- quent thoracic segments bear dorsal notopodia and ing to a widely accepted general model of chaetal ventral neuropodia (Fig. 1A). The thoracic notopo- replacement, formation of new chaetae occurs at a dium bears two clusters of capillary chaetae: a single special formative site at one end of the chaetal row, semicircle of 8–10 narrowly hooded capillary chae- while degeneration takes place at a degenerative site tae in the superior (dorsalmost) cluster, and two located at the opposite end of the row (Pilgrim parallel transverse rows of 10–12 paleate capillary 1977; Duchene & Bhaud 1988; Hausen 2005; Hoff- chaetae in the inferior (ventralmost) cluster mann & Hausen 2007). Newly formed chaetae enter (Fig. 1A,C) (note that all chaetal terminology used the row and push the older ones toward the other is sensu Fitzhugh 1989). Each thoracic neuropodium end; the oldest chaetae are either gradually resorbed bears two parallel tori with 19–28 acicular uncini in (Thomas 1930; Pilgrim 1977) or are shed (Duchene the anterior row and 18–24 avicular uncini in the & Bhaud 1988; Hausen 2005). posterior row (Fig. 1A,C). Each abdominal notopo- The outlined chaetal replacement model implies dium bears one transverse row of 15–20 avicular un- that each chaetal row should contain both formative cini, and each neuropodium bears two parallel and degenerative sites. Formative sites are easy to transverse rows, each consisting of 7–12 broadly find, and chaetal formation (chaetogenesis) in anne- hooded capillary chaetae (Fig. 1B,D). lids has been extensively studied by both light Adults of P. reniformis were collected by scuba and electron microscopies (Bartolomaeus 1995; divers in June 2010 from Kandalaksha Bay near the Meyer & Bartolomaeus 1996; Bartolomaeus & Meyer White Sea Biological Station (66°340 N, 33°080 E). 1997; Hausen & Bartolomaeus 1998; Hausam & Worms were cut into 2–4 pieces with scissors and Bartolomaeus 2001; Bartolomaeus 2002; Hausen placed into aquaria with running seawater to allow 2005). However, degenerative sites have been regeneration. During regeneration, the branchial described only for Clymenella torquata (LEIDY 1855) crown, pygidium, and the first two thoracic chaeti- by Pilgrim (1977), Capitella capitata (FABRICIUS 1780) gers are formed de novo from the apical blastema, by Schweigkofler et al. (1998), and Owenia fusiformis while the remaining thoracic segments (typically DELLE CHIAJE 1844 by Meyer & Bartolomaeus (1996). 6–7) transform from the nearest 6–7 abdominal seg- It is not clear why degenerative sites are so elu- ments (a phenomenon known as morphallaxis). The sive. One possible explanation is that degenerative transforming segments lose abdominal-type chaetae, sites are difficult to observe because chaetae are lost which are replaced by new thoracic-type chaetae so quickly that observing degeneration of an indi- (Figs. 2, 3A–F). The reorganization takes about vidual chaeta is nearly impossible. Alternatively, the 100 d (Kolbasova et al. 2013). To establish the chaetal replacement model may not be universal, course of morphogenetic events following the para- and in some species, chaetal degenerative sites might podial reorganization, every 3–5d,5–8 worms were not form at all. To address this question, we exam- photographed and fixed for further examination ined morphogenetic events during chaetal degenera- using both scanning and transmission electron tion and replacement in the sabellid polychaete microscopy (Kolbasova et al. 2013). For scanning Pseudopotamilla reniformis (BRUGIERE 1789). Chaetal electron microscopy (SEM), the were fixed replacement as a result of thoracic region regenera- in 2.5% glutaraldehyde in PBS buffer with 1%

Invertebrate Biology vol. 133, no. 3, September 2014 Chaetal loss in polychaetes 263

Fig. 2. Schematic illustration of parapodial reorganiza- tion in Pseudopotamilla reniformis. A. Intact abdominal parapodium. B. At the 10th day after fragmentation, abdominal notopodia lose their uncini, and formative sites both in noto- and neuropodial chaetal sacs disinte- grate. C. At the 17th–20th day after fragmentation, new chaetal sacs and formative sites develop, and thoracic capillary chaetae and uncini arise from the chaetal sac. D. At the 25th–30th day after fragmentation, abdominal- type neuropodia are partially reduced. E. At the 50th– 70th day after fragmentation, abdominal-type neuropodia are reduced completely. F. At the 100th day after frag- Fig. 1. Parapodia of Pseudopotamilla reniformis. A. Tho- mentation, thoracic parapodia are fully formed. acc, racic parapodia. White arrows mark formative sites. a, abdominal-type capillary chaetae; au, abdominal-type un- anterior row of acicular uncini; ia, inferior cluster of pal- cini; fs, formative sites of chaetal rows; tcc, thoracic-type eate capillary chaetae, anterior row; ip, inferior cluster of capillary chaetae; tu, thoracic-type uncini. Arrows indi- paleate capillary chaetae, posterior row; s, superior cluster cate the direction of chaetal and uncinal row formation. of narrowly hooded capillary chaetae; tcc, thoracic capil- lary chaetae; tu, thoracic uncini. B. Abdominal parapodi- a. White arrows mark formative sites. a, anterior row of and transmission electron microscopies (TEM), broadly hooded capillary chaetae; acc, abdominal capil- worms were fixed in 2.5% glutaraldehyde in 1 lary chaetae; au, abdominal uncini; p, posterior row of 0.1 mol L PBS buffer for 3–6 h (depending on broadly hooded capillary chaetae. C. Schematic diagram their size), postfixed with 1% osmium tetroxide of a thoracic parapodium. Black arrows mark formative (OsO4 1%) for 1 h, dehydrated in an ethanol- sites. D. Schematic diagram of an abdominal parapodium. acetone series, and embedded in Epon. Semi-thin Black arrows mark formative sites. sections (900 nm) were cut with Dupont MT 5000 and LKB-III microtomes, stained with a mixture of OsO4, dehydrated in an increasing ethanol series, methylene blue and toluidine blue, and examined critical point dried with CO2 in a Hitachi HCP-2, with compound microscopes. Digital images were mounted on aluminum stubs, and sputter-coated captured with a Leica DFC 290 camera and Leica with a Au-Pd mixture using an Eiko IB-3 Ion Application Suite software. Ultra-thin sections Coater. Specimens were examined using a Cam Scan (50 nm) were cut with LKB-III and Leica UC 6 mi- S-2 scanning electron microscope. For both light crotomes, collected on formvar-covered single slot

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Fig. 3. Stages of abdominal parapodial reorganization in Pseudopotamilla reniformis. A. An intact abdominal parapo- dium, with the dorsal notopodium bearing avicular uncini and the ventral neuropodium bearing capillary chaetae. B.At the 10th day after fragmentation, the parapodium is in its initial stage of reorganization. The notopodium has shed most of its uncini. C. At the 16th–20th day after fragmentation, the parapodium is in the early stages of thoracic-type notopo- dium (capillary chaetae) development. D. At the 25th–30th day after fragmentation, abdominal-type neuropodia (which contain capillary chaetae) are reduced. E. At the 50th–70th day after fragmentation, abdominal-type neuropodia (which contain capillary chaetae) are lost completely. F. A fully formed thoracic parapodium that has undergone re-organiza- tion from the abdominal pattern of chaetal distribution to that of a thoracic parapodium. acc, abdominal-type capillary chaetae; tcc, thoracic-type capillary chaetae; tu, thoracic-type uncini. Arrows mark formative sites. copper grids, stained with uranyl acetate and lead convenient for observations because they were smal- citrate, and examined with a JEOL JEM-1011 elec- ler and were replaced more rapidly. Therefore, only tron microscope. Digital images were captured with the degeneration of uncinal follicle cells is described a Gatan ES500W Erlangshen camera and SIS Mega in detail below. View Software. Morphology of parapodia and chaetal sacs Results Structure of thoracic parapodia. Each thoracic The loss of both of capillary chaetae and uncini parapodium had two chaetal sacs, one notopodial and occurred in the same way, but uncini were more one neuropodial. The chaetal sac of the notopodium

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the epithelium, never extending to the surface; they were positioned in a regular fashion between uncini along the uncinial row (Figs. 1D,5A–D). Both uncini and inner chaetae had ventral formative sites that were clearly visible as dark cells filled with electron- dense stained vesicles (Fig. 5B). Degenerative sites were not found in the notopodial chaetal sac; cells at the ventral edge of a chaetal sac were similar to the other cells in the chaetal sac, except for formative site cells (Fig. 5A). The neuropodial sac of capillary chae- tae was located on the ventrolateral body wall (Fig. 1B,D). The formative sites of these chaetal rows were dorsal; we found no degenerative sites in the neuropodial chaetal sac (Fig. 1B,D).

Organization of abdominal notopodia chaetal sacs. Each intact uncinal chaetal sac was organized as an invagination of the epithelial basal lamina, forming a groove where uncinal follicles were situ- ated (Figs. 4B, 5A,B, 6). Uncini were formed in the formative site situated in the cavity on one end of the chaetal sac. Each uncinus was surrounded by its own follicle. Five follicular cells—the chaetoblast, two follicle cells, and two associated epidermal cells—were visible in transverse sections of the folli- cle of a fully differentiated uncinus (Figs. 4B, 6). The chaetoblast, which was found in the basal part Fig. 4. Schematic illustration of uncinal chaetal sacs in of the uncinal follicle, was extremely ramified and parapodia of Pseudopotamilla reniformis. A. Thoracic interdigitated with folds of adjacent follicle cells, neuropodium. B. Abdominal notopodium. a, anterior row and its cytoplasm stained intensely owing to numer- of acicular uncini; au, abdominal uncini; bl, epithelial ous intermediate filaments attaching the uncinus to basal lamina; c, cuticle; ch, chaetoblasts; ec, associated the basal lamina. epidermal cells; fc, follicle cells; if, intermediate filaments; Two follicle cells were adjacent to the apical part m, musculature of the chaetal sac; p, posterior row of of chaetoblast in the middle part of the follicle; avicular uncini; tu, thoracic uncini. together, these covered the shaft of the uncinus (Figs. 4B, 6). These follicle cells formed distinctive extended deep into the epithelium and contained folds (Figs. 4B, 6), and their cytoplasm was paler three chaetal rows having ventral formative sites than that of the chaetoblast. Intermediate filaments (Fig. 1A,C). In contrast, the neuropodial uncinal inside the follicle cells attached the uncinus both to sac was not deep and contained two rows of uncini the basal lamina and to the musculature of the chae- (Figs. 1A,C, 4A). Darkly staining cells clearly indi- tal sac (Figs. 4B, 6). The apical part of the follicle cated dorsal formative sites in both uncinal rows. consisted of two specialized epidermal cells sur- No signs of degenerative sites, such as broken chae- rounding the neck-like region of the uncinus tae or a high density of lysosomes inside follicle cell (Figs. 4B, 6). Unlike other epidermal cells, these had cytoplasm, were found in either thoracic or abdomi- a thick cuticle forming a ring-like region around the nal chaetal sacs. uncinal shaft (Figs. 4B, 6). Each inner chaeta had its own follicle that con- Structure of abdominal parapodia. Each abdomi- sisted of two cells, a ramified darkly stained chaeto- nal parapodium had a notopodial and a neuropodial blast and a large follicle cell (Fig. 5C,D). chaetal sac. The notopodial chaetal sac was located on the lateral body wall and gave rise to a row of Chaetal replacement during regeneration uncini and a row of inner chaetae (Figs. 1B,D, 4B). The inner chaetae were small (up to 15 lm long and Chaetal replacement in a notopodium. One to five 2 lm in diameter), and were completely embedded in days after fragmentation (n=6 individuals), there

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Fig. 5. Ultrastructure of the notopodial chaetal sac in an abdominal parapodium of Pseudopotamilla reniformis. A. Dorsalmost part of the chaetal sac. B. Ventralmost part of the chaetal sac, with the formative site clearly identifiable by electron-dense stained cells. C. A parasagittal section through the notopodial chaetal sac showing inner chaetae. D. A parasagittal section through the notopodial chaetal sac showing a follicle of an inner chaeta. Drawings indicate orientation and planes of sections. acc, abdominal capillary chaetae; au, abdominal uncini; c, cuticle; ch, chaetoblast; fc, follicle cell; fs, formative site; ic, inner chaetae; m, musculature of the chaetal sac; n, nucleus of follicle cell; s, plane of parasagittal section. White arrows point to epithelial basal lamina. were no external signs of chaetal reorganization ventralmost uncini from the main notopodial tori (Fig. 3A). During the 7th–10th days (n=8), the noto- were lost first, but the most recently formed uncini, podial uncini of transforming segments fell out very still located near the formative site in the dorsal part quickly, usually within 18–20 h (Fig. 3B). The of the row, remained in the notopodia several hours

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Fig. 6. Ultrastructure of an abdominal uncinial follicle of Pseudopotamilla reniformis. au, abdominal uncinus; c, Fig. 7. Abdominal notopodium reorganization in Pseudo- cuticle; ch, chaetoblast; ec, associated epidermal cells; fc, potamilla reniformis, 10 d after fragmentation. A. Abdom- follicle cells; m, musculature of chaetal sac; n, nuclei; if, inal notopodium after loss of all notopodial uncini. intermediate filaments. Arrows indicate epithelial basal B. Tissue of the notopodial chaetal sac. C. Degeneration lamina. of inner chaeta follicles. acc, abdominal capillary chaetae; bl, basal lamina; c, cuticle; cs, chaetal sac; ic, inner chae- longer (Fig. 3B). The spot where the uncini fell out tae; if, intermediate filaments; n, nucleus of the follicle cell; tcc, thoracic capillary chaetae. was marked by a deep groove (Fig. 3B). During this period, a new sac of thoracic capillary chaetae started to form. It emerged near the ventral side of emerged on the notopodial surface. On the 16th– the notopodial sac as a small invagination of the 20th day after the fragmentation (n=6), the old unci- basal lamina filled with the darkly stained cells nal sac disappeared completely. The new chaetal sac characteristic of new formative sites. During the produced 4–5 narrowly hooded capillary chaetae in 11th–15th days (n=5), a number of large electron- the superior row, 1–2 paleate capillary chaetae in transparent vesicles appeared in chaetal sac cells, the the anterio-inferior row, and 3–4 paleate capillary cells membranes disappeared, and the nuclei became chaetae in the posterio-inferior one (Fig. 3C, pale and disintegrated (Fig. 7A–C). The intermedi- Fig. 8A). ate filaments were easily seen, and the muscular sys- The podial bulb and the musculature of the new tem of the chaetal sac remained present in the chaetal sac appeared by the 21st–30th day post-frag- parapodial tissue (Fig. 7C). mentation (n=4). During this period, notopodia The new sac of capillary chaetae was much deeper already bore 5–6 narrowly hooded chaetae in the in the parapodium than the old uncinal sac, because superior row, 2–3 paleate capillary chaetae in the capillary chaetae are much longer than uncini. Three anterio-inferior row, and 3–4 paleate capillary chae- formative sites of three new chaetal rows were local- tae in the posterio-inferior row (Fig. 3D). At the ized under the old uncinal sac, and as a result, the 50th day (n=2) post-fragmentation, there were 6–8 new thoracic chaetae grew through the degenerating narrowly hooded chaetae in the superior row, 3–4 uncinal chaetal sac (Fig. 7A). First, one or two nar- paleate capillary chaetae in the anterior row, and 4– rowly hooded capillary chaetae from the superior 5 paleate capillary chaetae in the posterior one. The row appeared. A day later, one paleate capillary musculature of the notopodia continued to develop, chaeta from the anterio-inferior row and one paleate and the podial bulb enlarged significantly (Fig. 3E). capillary chaeta from the posterio-inferior row Subsequently, the number of chaetae in notopodia

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one. In semi-thin sections, the newly forming uncini become visible 15–18 d after fragmentation (n=3) (Fig. 8A). At this point, the new thoracic uncinal sac started to elongate toward the old abdominal chaetal sac (Fig. 8A). During the 16th–20th days post-fragmentation (n=6), new uncini appeared at the body surface (Fig. 3C). Avicular uncini appeared 1–3 d later than acicular ones. During the 25th–30th days (n=8), both types of uncini formed two more or less parallel rows, each consisting of 4–7 uncini (Fig. 3D). As the uncinal rows became longer, a new uncinal sac extended ventrally and fused with the old neuropo- dial sac (Fig. 8B). At the same time, the old neuropo- dial sac became smaller, and its muscular system began to be resorbed (Fig. 8B). This process occurred around the 50th day post-fragmentation (n=2). The last remaining capillary chaetae were shed from the neuropodium during the 60th–80th day after fragmentation (n=8). Finally, after 90 d (n=5), the old neuropodial chaetal sac disappeared com- pletely and the former abdominal parapodium (both noto- and neuropodium) reached the adult thoracic condition (Fig. 3F).

Follicle cell degeneration during chaetal regeneration During the 1st–3rd day after fragmentation (n=3), there were no signs of degeneration in follicle cell cytoplasm; their membranes remained distinct and easily visible (Fig. 9A). The first changes in the folli- cle cell cytoplasm occurred during the 4th–5th day (n=2); during this time, numerous lysosomes appeared in the cytoplasm, and cell membranes began to disintegrate (Fig. 9B). Simultaneously, small elec- Fig. 8. Reorganization of a neuropodium in Pseudopo- tron-transparent vesicles appeared in follicle cells. tamilla reniformis. A.12–20 d after fragmentation. B.25– Two days later, at the 6th or 7th day post-fragmen- 30 d after fragmentation. acc, abdominal capillary = chaetae; c, cuticle; cs, chaetal sac; fs, formative site; tcc, tation (n 2), follicle cell membranes were indistinct thoracic capillary chaetae; tu, thoracic uncini. Arrows (as observed by TEM), but the cytoplasm was still indicate epithelial basal lamina. distinct. The number of lysosomes increased (Fig. 10A). Although cell membranes disintegrated, gradually increased; completely reorganized notopo- the uncinal intermediate filament system was still dia were present by the 90th–120th day post-frag- present in the cytoplasm. On the 7th–10th day post- mentation (n=5) (Fig. 3F). fragmentation (n=1), uncini fell out, electron-trans- parent vesicles enlarged considerably, and the Chaetal replacement in a neuropodium. Formation follicle cell cytoplasm looked torn and porous of new neuropodia took longer than that of notopo- (Fig. 10B). The intermediate filament system disap- dia. On the 10th–15th days after fragmentation peared around the 10th–12th day post-fragmenta- (n=5), when the first new notopodial capillary chae- tion (n=3). tae appeared on the body surface, the neuropodium showed no external changes (Fig. 3B). During this Discussion period, a new neuropodial chaetal sac with forma- tive sites of two uncinal rows appeared between the We have shown that parapodial reorganization new notopodial chaetal sac and the old neuropodial in Pseudopotamilla reniformis proceeds via disinte-

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Fig. 9. Degeneration of follicle cells in Pseudopotamilla reniformis. A. Two days after fragmentation. B. Four days after fragmentation. Diagrams indicate the plane of sections. au, abdominal uncinus; bl, epithelial basal lamina; ch, chaetoblast; cm, cell membrane; ec, associated epidermal cells; fc, follicle cells; if, intermediate filaments; ly, lysosomes; m, musculature of the chaetal sac. gration of the old chaetal sacs and formation of suspect that all formative sites disappear along new chaetal sacs. In both old abdominal-type and with the old abdominal chaetal sac and the new new thoracic-type parapodia, notopodial formative formative sites form in a new thoracic sac sites are ventral and neuropodial formative sites (Fig. 2). are dorsal. New thoracic chaetae (or uncini) fol- During uncinal sac disintegration, all uncini low the abdominal ones in their course of forma- in the row can fall out simultaneously and thus tion (the row elongates toward the dorsal body chaetal loss is not associated with a degenera- side in a notopodium and toward the ventral body tive site located in a particular area of the sac. side in a neuropodium). It is unclear whether the Our investigation allows subdivision of the pro- notopodial formative site persists, triples, and cess of chaetal degeneration into three main begins to produce a new type of chaetae during periods. Initially, numerous lysosomes and the parapodial reorganization, or if it is replaced small electron-transparent vesicles appear in the by three new formative sites. It is also unclear if cytoplasm of follicle cells, indicating the begin- two old neuropodial formative sites are replaced ning of membrane disintegration. The next step by two new uncinal formative sites, or they switch involves disintegration of the follicular cell to formation of two uncinal rows. Because of sig- membranes. The final stage is complete dissolu- nificant morphological and topological differences tion of follicle cells, resulting in chaetae being between the uncinal and capillary chaetal sacs, we shed from the parapodium.

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Fig. 10. Degeneration of follicle cells in Pseudopotamilla reniformis. A. Five days after fragmentation. B. Seven days after fragmentation. Diagrams indicate the plane of sections. au, abdominal uncinus; bl, epithelial basal lamina; if, intermediate filaments; ly, lysosomes; m, musculature of the chaetal sac.

However, a totally different type of chaetal reported for degenerative sites in rows of hooked degeneration was described in rows of acicular chaetae of Capitella capitata by Schweigkofler et al. uncini of the maldanid polychaete Clymenella torqu- (1998) and Owenia fusiformis by Meyer & Bartolo- ata (Pilgrim 1977). In this species, chaetae are not maeus (1996). It suggests that in all three species shed, but chaetal degradation occurs by formation (the capitellid C. capitata, the oweniid O. fusiformis, of “waste sacs” at degenerative sites where old and and the sabellid P. reniformis), at the cellular level, broken chaetae are deposited into the coelom and chaetal degeneration takes place in the same manner later completely destroyed by coelomocytes. Such a regardless of the presence or absence of functioning mode of chaetal loss seems unusual because it degenerative sites in parapodia. Therefore, the pres- means that the old chaetae have to pass through ence of numerous lysosomes is a universal marker the basal lamina and musculature of the chaetal of chaetal loss 2–3 d before it occurs, and so may sac. It is possible that similar chaetal resorption serve as a histological indicator of chaetal degenera- takes place during the degeneration of inner chae- tion in a wide range of . Such a marker tae in P. reniformis (Figs. 7B,C). Although we have makes it possible to detect signs of chaetal degenera- not specifically examined the process of chaetal tion whether they are restricted to specialized degen- replacement in intraepithelial chaetae, it seems that erative sites, or occurring anywhere along the they are resorbed inside chaetal sacs, along with chaetal row. follicle cells, at the same time as parapodia get In the parapodia of intact (non-regenerating) indi- reorganized. viduals of P. reniformis, TEM shows no evidence of The presence of numerous large lysosomes indi- chaetal degeneration, such as numerous lysosomes, cating the breakdown of old chaetae was also electron-transparent vesicles, or empty follicles.

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Scanning electron microscopy of the parapodia of of anterior chaetigers are replaced by capillary chae- intact adults of P. reniformis shows that both smal- tae; thus, the position of hook-bearing chaetigers ler juvenile-type and larger adult-type chaetae and depends on worm size (Tzetlin 1990). Similarly, in uncini are present. The adult uncini are positioned the spionid Dipolydora concharum, capillaries close to the formative sites, whereas the juvenile- develop between hooks in new segments, and are type chaetae are situated at the opposite end of the lost as the worm grows. Therefore, capillary chaetae uncinal row. This situation is a result of a row for- are always present only in a few newly developed mation, when new uncini develop in the formative posterior-most chaetigers (Radashevsky & Fauchald site and are gradually displaced toward the opposite 2000). In another spionid, Scolelepis squamata, the end of the row as the parapodium grows (Figs. 2, abdominal neuropodia initially bear a row of capil- 3). During parapodial reorganization in P. renifor- lary chaetae and a row of hooks, but the capillary mis, capillary chaeta formation takes about 3–7d, row degenerates before its formation is complete, while uncini form in 2–5 d (considering that capil- leading to a single transverse row of hooded hooks lary chaetae continue their formation 1–2 d after in anterior abdominal neuropodia (Hausen & Barto- they project above the body surface, unlike uncini, lomaeus 1998). Thus, in Lumbrineridae and Spioni- which complete their formation below the body sur- dae, one chaetal row replaces another row inside the face). The presence of juvenile-type uncini in the common chaetal sac. Such a replacement requires parapodia of intact P. reniformis, as well as the reorganization of one formative site type into the absence of any histological signs of chaetal degener- other, because a formative site produces only one ation, may suggest that even if such uncini get type of chaetae. Usually, when a chaetal row con- replaced as the grows, this replacement sists of two alternating types of chaetae, it suggests occurs very slowly. This would make it difficult to presence of two closely spaced chaetal rows with pinpoint the moment of chaetal replacement using two different formative sites. Therefore, we propose SEM and histological methods. On the other hand, that in abdominal notopodia of P. reniformis, there the life span of P. reniformis is unknown, and the are two separate formative sites that produce chae- rate of formation of both capillary chaetae and tae in rotation and give rise to two different rows. uncini is not uniform, as during regeneration 2–3 For the examples of Lumbrineridae and Spionidae new chaetae from a row appear simultaneously, mentioned above, it is not clear whether the replace- after which chaetal formation decelerates. Therefore, ment of an old chaetal row occurs through a degen- to assess the rate of chaetal replacement, one also erative site, or if all chaetae in the row fall out needs to compare the relation between the juvenile simultaneously. Chaetal replacement clearly takes and adult type of chaetae (or uncini) in worms of place without involvement of degenerative sites dur- different ages. ing rapid parapodial reorganization, when chaetae Having juvenile and adult chaetae in one chaetal are shed by entire chaetal sacs, as described here for row has been described in the terebellids Eupolymnia P. reniformis during regeneration. Similarly, rapid nebulosa (MONTAGU 1818), Lanice conchilega (PAL- chaetal replacement was also observed in nereidids LAS 1766), Neoleprea streptochaeta (EHLERS 1897), during epitokal transformation as described for € Nicolea zostericola ORSTED 1844, Pista cretacea Perinereis cultrifera (GRUBE 1840) by Clark (1961), (GRUBE 1860), Pista cristata (MULLER€ 1776), and and in P. reniformis during asexual reproduction Thelepus setosus (QUATREFAGES 1866) (Bhaud 1988; (Kolbasova et al. 2013). Parapodial reorganization Duchene & Bhaud 1988). In these studies, the capil- takes place as a result of induced post-traumatic lary chaetae and uncini varied in size and shape regeneration in the sabellids Branchiomma nigromac- with age (Bhaud 1988; Duchene & Bhaud 1988), but ulata (BAIRD 1865), melanostigma (SCHMAR- the observed slight variation was only noticeable in DA 1861), Sabella pavonina (SAVIGNY 1822), Bispira comparison with chaetae within the same row. In all volutacornis (MONTAGU 1804), Megalomma vesiculo- these cases, slow and gradual chaetal replacement sum (MONTAGU 1815), Branchiomma bombyx (DAL- most likely involves a degenerative site, as described YELL 1853), and Potamilla torelli (MALMGREN 1866) for C. capitata and O. fusiformis (Meyer & Bartolo- as described by Berrill (1931, 1977, 1978), Berrill & maeus 1996; Schweigkofler et al. 1998). Mees (1936a,b), and Licciano et al. (2012). The More complicated types of age-related chaetal same happens during spontaneous asexual reproduc- replacement are found in some Lumbrineridae and tion in serpulids Filograna BERKELEY 1835 and Spionidae. In the chaetigers of Lumbrinereis fragilis Salmacina CLAPAREDE 1870 (ten Hove 1979; Nishi & (Lumbrineridae), hooks appear more posteriorly in Nishihira 1994; Pernet 2001; ten Hove & Kupriya- older than in younger specimens. With age, hooks nova 2009). In the sabellids B. nigromaculata,

Invertebrate Biology vol. 133, no. 3, September 2014 272 Kolbasova, Tzetlin, & Kupriyanova

Potamethus elongatus (TREADWELL 1906), and several ———— 1977. Functional morphology and development of other unnamed “dwarf” sabellids, such parapodial segmental inversion in sabellid polychaetes. Biol. Bull. reorganization was observed during juvenile devel- 153: 453–467. opment (Berrill 1977). Although none of the above ———— 1978. Induced segmental reorganization in sabellid – studies addressed the type of chaetal loss, we worms. J. Embr. Exp. Morph. 47: 85 96. Berrill NJ & Mees D 1936a. Reorganization and regener- hypothesize that during parapodial reorganization ation in Sabella. I. Nature of gradient, summation and in Sabellida in general, chaetal replacement proceeds posterior regeneration. J. Exp. Zool. 73: 67–83. without involvement of degenerative sites. ———— 1936b. Reorganization and regeneration in Sabel- In conclusion, we suggest that chaetal degenera- la. II. The influence of temperature. III. The influence tion and loss in polychaetes can occur either of light. J. Exp. 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