Achaeta piti Bittencourt, 1974

Schmelz, Rüdiger M., Collado, Rut & Römbke, Jörg, 2008, Mata Atlântica enchytraeids (Paraná, Brazil): The genus Achaeta (Oligochaeta, Enchytraeidae), Zootaxa 1809, pp. 1-35 : 4-9

Achaeta piti Bittencourt, 1974

Figure 2 , Tables 1 , 4

Achaeta piti Bittencourt, 1974: 369 –371, Figs 1 –10

Anachaeta eiseni Vejdovský, 1878 — Michaelsen 1889a: 47 (partim, non Vejdovský, 1878) Collected specimens: MZUSP #1362, 8 spms (6 submature, 2 juvenile) from Cachoeira Natural Reserve, Paraná, Brazil, May 2003 (3 spms), March 2004 (3 spms), October 2004 (2 spms). MZUSP #1363, three specimens, one fully mature, from urban parkland in Curitiba, close to entrance of Department of Soils and Agricultural Engineering, Federal University of Paraná (UFPR), Section Agrosciences Agrarias, October 2004. All specimens as stained whole mounts. Further 10 specimens, investigated in vivo, not conserved.

Type series: MZUSP #277: 27 specimens, holotype and paratypes, Brasil, São Paulo, Osasco, coll. Bittencourt, VIII 1973. Of these, 25 spms preserved in 10% formol, and 2 spms in sections on slides. Formol-preserved spms: 13 fully mature, 10 submature (clitellum not or not fully developed, spermathecae and male reproductive organs present), 2 juvenile. Specimens were dehydrated and whole-mounted for this study, later retransferred to ethanol. Sections: specimens without posterior end, fully mature, in transverse sections on 5 and 6 slides, respecively.

Further reference material: ZMH V #458: 10 specimens, " Achaeta eiseni Vejd. ", Hamburg, Botanical Garden, flower bed, leg. et det. Michaelsen (Michaelsen 1889a: 47).

Description of new material: Living specimens ca. 5 mm long and 0.13 mm wide, up to 0.15 mm under coverslip. Preserved specimens 4 mm long and 0.1-0.14 mm wide (types: 3.5-5 mm long, 0.14-0.19 mm wide; ZMH material: 4.5–6.5 mm long, up to 0.25 mm wide). Segment number (16)-31-36 (N=8, types not included).

Pyriform epidermal glands ( Fig. 2 A) in 3 pairs per segment, one dorsal, one lateral, one ventral, all inserting in the posterior region of a segment, i.e. closer to respective posterior septum than to anterior septum; dorsal and lateral glands at the same transverse level, ventral glands inserting more posteriorly. Lateral glands inserting dorsally of lateral line (distance ca. 15 µm, fix), ventral glands inserting in longitudinal line of nephridial pores. Gland size variable, dorsal glands largest, length up to 1/3 of worm body diameter in anterior segments, smaller posteriorly; first pair at II, present also at XI in clitellar region; glands filled with vesicles. Lateral and ventral glands from III (laterals from II in 1 specimen of type series), absent at XI, about half the size of the dorsal glands, hyaline distally and granular proximally, and with small proximal knob (nucleus), larger in anterior segments. Lentiform epidermal gland cells seen in first, second and caudal segments of some whole-mounted specimens, apparently absent in rest of body.

Body wall of varying thickness, ca. 6–15 µm altogether, 1/20-1/10 of body diameter, cuticle ( Fig. 2 A,D) conspicuous, 2-5 (-10) µm thick, thicker dorsally than ventrally, varying among specimens; body wall and cuticle thicker in collected material than in type series and ZMH specimens, in the latter occasionally down to 4 μm. Ring muscles adjacent to one another, not superposed, 23–24 rings (rows) per segment, resulting transverse body wall striation conspicuous; longitudinal muscle layer well-developed, contributing to about half of the body wall thickness. Septa ( Fig. 2 A) 4/5-7/8 thickened.

Head pore on prostomium. Frontal prostomial epithelium ( Fig. 2 A) unequally thick, with two recesses ( Fig. 2 A: 15), one dorsally below head pore, one ventrally close to mouth opening. No elongate prostomial papillae. A few prostomial muscles present. Brain ca. twice as long as wide, about 80 μm long (fix) (up to 155 μm long and 77 μm wide in original description), anteriorly convex, posteriorly rounded or truncate, sides roughly parallel or slighty converging anteriad. Ventral nerve cord ( Fig. 2 A) ganglionated, suboesophageal ganglion in II–IV, undivided, subsequent ganglia segmental and separate. Post-pharyngeal bulbs present, 2 pairs, inner pair on oesophagus, outer pair on afferent fascicles of pharyngeal glands.

Oesophageal appendages ( Fig. 2 A,B) in IV and V; a pair of large ovoid bodies dorso-laterally in V, directly before pharyngeal glands; each body with a meandering canal of its own in IV (viv); opening into oesophagus not seen. Pharyngeal glands ( Fig. 2 A,B) in IV, V, VI; all united dorsally, dorsal connection widest in IV, narrowest in VI; glands in all three segments more or less U-shaped (top view), all with primary ventral lobes, these largest in IV; no secondary gland lobes. Posterior origin of dorsal blood vessel ( Fig. 2 A) in VII-1 / 2 VIII. Chloragocytes from VII, with single cells occasionally from V or VI; granula dark-grey-green in collected material. Gut dilatation gradual in VIII, abrupt in strongly contracted, preserved material. Pars tumida of midgut seen in four specimens, here circumferal, not constricted to ventral region, from XX-XXIV(XXV) in specimens with more than 30 segments (N=2), and from XII–XIV in juveniles with 17 segments (N=2); epithelium conspicuous by brownish refractile vesicles, not always elevated, not distinguished in two specimens; not scrutinized in the types.

Preclitellar nephridia ( Fig. 2 A,C) in two pairs, at 7/8 and 8/9, anteseptale about as long as wide, postseptale usually bent forward, gradually narrowing towards porus, altogether ca. 4x as long as anteseptale, no terminal vesicle. Postclitellar nephridia few in number (e.g., first pair at 18/19), shape and size the same as preclitellar nephridia, but usually straight.

Coelomocytes small, 15–18 µm long (19 µm long in original acccount), slightly longer than wide, flat and disc-like, numerous or scarce; cells with conspicuous vesicles, or vesicles blurred, irregular, pale, cells somewhat darker than coelom, brown in two specimens ( MZUSP #1363). Pygidium not longer than wide, anal muscles not strongly developed.

All reproductive organs except spermathecae shifted one segment forward: testis and sperm funnels in X, ovary, vasa deferentia and male pores in XI.

Clitellum ( Fig. 2 D) in XI and XII, short, extending over little more than one segment length, beginning right behind or at intersegmental furrow of 10/11 and including about 8 ring muscle rows of XI; open dorsally and ventrally, cells in longitudinal and transverse rows. On each side dorso-laterally two large, elongate, baguette-like packages of hyalocytes, extending over entire clitellum length (border cells excepted), deeply projecting into body cavity when fully developed; each package lined with longitudinal rows of flat granulocytes, one to the left, one to the right. Ventro-laterally only granulocytes, in 3 inconspicuous longitudinal rows. At anterior and posterior end of clitellum, 3–5 transverse rows of hyaline border cells, respectively. Gland-cell free area wide dorsally, narrowed in fully mature specimens, ventral clitellum-free area about as wide as distance between male pores, narrower at anterior and posterior end (border cells).

Seminal vesicle absent; cysts fill dorsal half of X. Spermatozoa ca. 44 µm long, heads 15-20 µm long (viv); heads 16 µm long in type specimens. Sperm funnel ( Fig. 2 D) cylindrical, slightly flattened, ca. half as long as body diameter, more than 3x as long as wide in fully mature specimens (110 µm: 30 µm, fix), shorter in submatures, oval in cross-section; collar distinct, flat, not wider than funnel body; funnel body with large vesicles, sperm in dense aggregations on top of collar. Vas deferens in numerous dense or in several wide irregular coils, diameter 4–5 µm (fix), same diameter throughout. Male pores in XI, widely separate, on body surface, surrounded by glandular body and muscles; glandular body ( Fig. 2 D) compact, flat (ca. 25 µm long, 22 µm wide, fix), subdivided into an anterior and a posterior lobe; copulatory muscles as dense network, distinguishable only in sections, allowing papillate protrusion of male pores. Further accessory glands near male pore absent. No subneural glands.

Spermathecal ectal pores lateral ( Fig. 2 A). Ectal duct short, canal lined by cuticle. Ectal sperm-containing dilatation of ampulla asymmetrical, diverticulum-like, usually oriented ventrally (dorsally on one side in one animal). Ampulla extending into VI or VII, connecting tube as wide as ectal duct, ental reservoir small, ca. twice as wide as connecting tube, with irregularly thick epithelium. One mature egg at a time, extending over 2 segments when fully developed.

Remarks. Comparison with original description and type series: The forward shift of sexual organs and the number of segmental pyriform glands disagree with the original description. According to Bittencourt (1974), there are dorsal and ventral pyriform glands (termed "sacos setígeros" in the text written in Portuguese), but lateral glands are not mentioned, which means 4 instead of 6 glands per segment. Furthermore, the reproductive organs in the clitellar region are described as being in the usual position, with testis in XI, egg in XII and clitellum at XII-1 /2 XIII. Since these two characters are among the principle traits to distinguish species in the genus, we first ascribed our material to a new species, closely related to A. piti . Reinvestigation of the type series, however, revealed the errors of the original description. All type specimens have a pair of dorsal, lateral and ventral pyriform glands, and there is a forward-shift of sexual organs in all mature and submature formol-preserved specimens. (The latter trait was—for obvious reasons—not seen in the two wholemounted juveniles and could not be made evident in the two transversely sectioned specimens.)

Most of the other traits in our material correspond with the original account, notable are general size and segment number, presence of pyriform glands, brain shape, presence, shape and size of the oesophageal appendages, details of the pharyngeal glands, position of first preclitellar nephridium, inward-projection of four longitudinal rows of clitellar gland cells laterally, general shape of the male reproductive system, and shape of the spermatheca. Several details have been added to the original description here; notable are location of spermathecal ectal pores, the number and location of thickened septa and preclitellar nephridia, and details of the clitellum, e.g., presence and distribution of granulocytes. Some taxonomically unimportant differences of the original description concern maximum body diameter (originally given as 252–266 µm and probably measured on flattened specimens during live investigation), relative length of pyriform glands, and brain size. The sperm funnel is cylindrical and not ovoid (comp. Bittencourt 1974, Fig. 10) in all specimens, the types included. The clitellar hyalocytes are said to coincide in position with the dorsal pyriform glands, but the latter are positioned more mid-dorsally, which is demonstrated by their presence in the clitellar segment (comp. Fig. 2 D).

New species diagnosis, comparison with other species: Type series and collected material are remarkably congruent in all characters, the thickness of body wall and cuticle excepted. Two specimens had brown coelomocytes. Achaeta piti appears to be a morphologically homogeneous species. All specimens, living or preserved, and juveniles included, can be identified beyond doubt by a set of six characters: (1) six pyriform glands per segment, one dorsal, lateral and ventral pair, respectively; (2) oesophageal appendages large, in IV–V; (3) pharyngeal glands all united dorsally, without secondary ventral lobes; (4) septa 4/5-7/8 thickened; (5) origin of dorsal blood vessel in VII-1 / 2 VIII; (6) two pairs of anterior nephridia, at 7/8 and 8/9. Mature specimens are further unmistakably recognized by the peculiarities of clitellum, male reproductive system and spermathecae, see the description for details.

Achaeta piti is now the second species in the genus with six pyriform glands per segment; this trait was so far only known for A. aberrans Nielsen & Christensen, 1961 . The glands are exactly in the same segmental position in both species (Graefe, Schmelz, unpublished observations). A. piti resembles A. aberrans in a few more characters: oesophageal appendages present, all pharyngeal glands connected dorsally, spermathecal ectal openings lateral. Differences of A. aberrans from A. piti are, among others: (1) segment number lower (20-23), (2) oesophageal appendages very small, (3) preclitellar nephridia at 6/7, 7/8, (4) dorsal blood vessel originating in VI, (5) reproductive organs in usual position, not shifted one segment forward, (6) clitellum without large, elongate, dorso-lateral packages of hyalocytes, (7) spermathecae without ectal asymmetry. A forward shift by one segment of sexual organs (except the spermathecae) occurs also in A. abulba Graefe, 1989 , A. bibulba Graefe, 1989 , A. pannonica Graefe, 1989 , A. diddeni Graefe, 2007 , and in A. hanagarthi sp. nov., described in this paper. These species are compared in Table 1 .

Further observations: A clitellum similar to the one in A. piti is very common in Achaeta , but not found outside the genus. The 'baguettes' vary considerably in size; they are inconspicuous in all submature and even in many mature specimens; it seems that the hyalocytes achieve their maximum volume only directly before the release of an egg—the granulocytes contribute little to the size increase of the 'baguettes'. The 'baguettes'

project deeply into the coelom, there is no outward bulging. The body wall muscles do not follow this inward bulge but rather leave clefts through which the 'baguette' cells penetrate into the coelom. This gives the impression as if the clitellar cells were located below the muscle layer. The longitudinal muscles are restricted here to narrow strands between the 'baguettes', close to the body surface; there are three strands on either side of the body wall, one ventrally of each the two baguettes, and one in the midst of the latero-ventral granulocyte layer; these strands spread out anteriorly and posteriorly of the clitellum to regain the continuous muscle cover across the worm diameter. The clitellar ring muscle strands in turn maintain their position, and their gaps are much narrower.

Register of type series: The extant type series differs in several instances from the respective details given in the original description. According to Bittencourt (1974: 369), the type series consists of 13 specimens, 8 of them mature, 5 juvenile, and the holotype is catalogued under #277, the paratypes under #227A(sic!). Furthermore, transverse and sagittal sections were made of an unspecified number of specimens. All material was deposited at the "Departamento de Zoologia do Instituto de Biociências da Universidade de São Paulo", now the Zoological Museum of the University of São Paulo. An inventory carried out by the first author in March 2004 and the scrutiny of the material as presented in this paper revealed the following: There are 25 formol-preserved specimens in a vial catalogued under #277 (which according to the number should contain only one specimen, the holotype); of these, 13 are fully mature, 10 are submature (without or with incompletely developed clitellum, spermathecae and male organs present), and two are juvenile. There is no vial with #227A in the Enchytraeidae collection (or #277A, the published number is probably a printing error). The slide collection contains two transversely sectioned specimens on 5 and 6 slides, respectively, unnumbered. This means: (1) There are more specimens in the type series than originally indicated. (2) It is not evident whether the sections are part of the type series. The original description is not clear on that point, either. If the sections are included, then the type series is incomplete since the sagittal sections mentioned in the original description appear to be lost. (3) The name-bearing type (holotype) is lost or cannot be identified--it may or may not be one of the 25 specimens in vial #277. The Code (ICZN 1999) to our knowledge provides no regulation for such a case. However, the whole problem is merely formal, since all specimens belong undoubtedly to one and the same species. Hence we assume for convenience that the type series consists of all specimens in vial #277 plus the two unnumbered transverse sections—the description makes use of these sections, comp. Fig. 1 and 4 in Bittencourt (1974) —and that the holotype is extant but unidentifiable, being one of the 25 formol-preserved specimens. Apparently, holotype and paratypes have been stored in one and the same vial, and a vial #227A (or 277A) has probably never existed. The type series consists then of holotype and 26 paratypes, 2 of them sectioned on slides.

ZMH material: More than a century ago, Michaelsen collected Achaeta specimens from a flower bed in the Botanical garden of Hamburg and identified them as Anachaeta eiseni Vejdovský, 1878 , a species with four pyriform glands per segment. (Nomenclatural note: Anachaeta Vejdovský, 1879 has been a transitory replacement name for the original Achaeta Vejdovský, 1878 , due to a presumed homonymy with Acheta (Hexapoda, Gryllidae ); however, the spelling is different and Michaelsen (1900) restored the old name). These specimens ( ZMH V #458), together with a single find from garden soil near Hamburg ( ZMH V #459, not investigated here), underly the record of A. eiseni from Northern Germany in Michaelsen (1889a, 1903). A description of the material was never published. The index card, written by Michaelsen himself, gives only his monograph ( Michaelsen 1889a) as reference, where the species is listed without description. In 1974 the material was reinvestigated by U. Graefe (in litt.) who discovered the erroneous identification and the presence of six segmental pyriform glands in the specimens. He kept the material for a future description and handed it to the first author in May 2007. The material is complete—the index card notes 10 specimens—and most characters are still distinguishable, although the material is not in optimal conditions. The specimens agree with our diagnosis of A. piti except that they are somewhat larger (length up to 6.5 mm, diameter up to 0.22 mm), which may be a storage artefact. Two specimens have a fully developed clitellum, the rest is submature or almost mature. The material ( ZMH V #458) was listed as missing in an inventory of the enchytraeid collection at the Zoological Museum in Hamburg ( Dózsa-Farkas 1997).

Figures

FIGURE 1. Location of the ' Reserva Natural Rio Cachoeira', study area and type locality of Achaeta hanagarthi, A. paranensis and A. singularis spp. nov.

FIGURE 2. Achaeta piti Bittencourt, 1974, specimens from the Cachoeira Natural Reserve. B is a freehand sketch from living specimens, the rest is from stained whole mounts. Bar = 100 µm, valid for A, C, D. A. Submature specimen, side view, anterior 9 segments, with thick body wall and dorsally thick cuticle (1). There are dorsal, lateral and ventral pyriform glands (2,3,4, respectively; only glands of left body side shown). The ventral glands insert more posteriorly than dorsal and lateral glands. The dorsal blood vessel (5) begins at 1 / 3 VIII in this specimen. The thickened septa in 4 / 5 – 7 / 8 are a constant feature of this species. 6: Oesophageal appendage; 7: pharyngeal gland; 8: postpharyngeal bulb; 9: brain; 10: suboesophageal ganglion; 11: ganglion of segment V. Perikarya are omitted here, they are as in Figs 3 B, 4 A. 12: nephridia; 13: intestine with chloragocytes; 14: spermatheca with ectal asymmetry; 15: recesses in frontal prostomial epithelium. B. Oesophageal appendage and pharyngeal glands, dorsal view, as seen in a living specimen. The two separately winding canals in IV, one for each glomerulus in V, were already mentioned in the original description. C. Postclitellar nephridia, side view. The organs lie in consecutive segments, one to the left, one to the right. D. Clitellum and male reproductive system, side view, specimen with thick body wall and cuticle. The dorsal pyriform gland of XI is drawn in (1). The clitellar border cells (2) extend farther ventrally than the granulocytes.