I ()6-171 S. Afr. J. Zoo!. 1998,33(3)

The morphology of the female reproductive structures of Penonyx excavatus (Oligochaeta)

Sophie A Reinecke Department of Zoology, University of Stellenbosch, Private Bag X1, Matieland, 7602 South Africa e-mail: [email protected] Rialet Pieters' Department of Zoology, Potchefstroom University for CHE, Potchefstroom, 2520 South Africa e-mail: [email protected]

Received 4 ,/\"'ovember /997; accepted 30 lv/arch /998

Perionyx excavatus (Oligochaeta) is a vermicomposting earthworm of which little is known regarding its repro­ ductive strategies. This is a second paper by the authors on the morphology of the reproductive structures in an attempt to reconcile the scanty and confusing literature on this topic and to investigate the possibility of self-fer­ tilization. The investigation was done by examining the gross anatomy and histology, using scanning and trans­ mission electron microscopy. The female reproductive system consists of three pairs of spermathecae in segments seven, eight and nine. Each spermatheca consists of one ampulla and four sessile diverticula. The spermathecae open separately but midventrally in the intersegmental groove anterior to the segments in which they occur. One pair of ovaries is found in segment 13. The ova are releas,:d into the .body cavity and ~re ~~I­ lected by the two ciliated ovum funnels, also in segment 13. The two short OViducts leading from the ovaries, JOin into a common oviduct. The common oviduct opens into the female opening, which is situated midventrally, anterior to the ring of body chaetae on segment 14.

*To whom corr~5pondence should be addressed

PeriUf1.H excavatus, an earthworm belonging to the family paper is concerned \vith the morphology of the female repro­ Megasculecidae, is utilised in vermicomposting (Neuhauser, ductive structures.

. Loehr & Malecki 1988) since it is a very prolific epigeic spe­ )

9 cies. It was initially described in 1872 by Perrier and is Material and methods 0

0 endemic to the southern regions of Asia (Gates 1972). Until 2 The specimens of P. excavatus used during the present study

d recently not much was known about the biology of P. excava­ were from a breeding stock kept in the laboratory of the e t Ills. Reinecke & Hallatt (1989) published information on a Department of Zoology at Potchefstroom University. This d grO\vth and cocoon production and the life cycle was ( stock originated from cucoons provided by Prof. Graff of o. r described by Hallatt, Reinecke & Viljocn (1990). Publica­ e Braunschweig (Germany). The breeding stock was main­ h s tions on the moisture requirements (Hallatt, Viljoen & Rei­ tained in an environmental control chamber at 25°C and 80% i l necke 1992) and the influence of wonn densiIY on cocoon b r.h. in a cattle manure substrate. u production (Reinecke & Reinecke 1994) also appeared.

P To obtain adult worms, cocoons were collected from the e Studies on the biology of this species have shown that both breeding containers and incubated in distilled water at 25°C. h t paired worms and single specimens, hatched and maintained One-day-old hatchlings were placed into 95 em; glass flasks y b in isolation, produced viable cocuuns. The question arose as in groups of three to eight individuals per flask containing a d to the strategy used by the isolated \vonns to enable them to e cattle manure substrate. This substrate was prepared by grind­ t n produce offspring. One possibility, that of self-fertilization as ing and sieving sun-dried, urine-free cattle droppings to a r postulated by Hallatt el ui. (1990), was examined by studying obtain a particle size of 500 11m. This particle size was found g

e the morphology of the reproductive organs. The aim was to to be favourable for the growth of P. excavulus (Reinecke & c n determine whether any physical connections or other mecha­ Hallatt 1989; Hallatt el ai. 1990). Distilled water was added e c i nisms might exist enabling spenn to reach the ova of the same to obtain a moisture content of 800/0 because this is the muis­ l

r \\lonn internally, resulting in self-fertilization. ture content at which these worms grow and reproduce best e d No comprehensive infonnation regarding the female repro­ (Hallatt el 01. 1990). n u ductive structures has been published and the descriptions As soon as clitellate worms could be obtained, from the

y presented by Bcddard (1886,1892), Edwards & Lofty (1972) a fourteenth day after hatching (Hallatt el ai. 1990), 25 speci­ w and Hanumante (1975) are in some instances contradictory. mens were prepared for dissection to determine the position e t Ovaries. ovum funnels and oviducts were not mentioned by a and number of repruductive organs and pores. Each worm

G Beddard (1886. 1892). Edwards & Lofty (1972) or Gates was rinsed in dlstiJJed \,,,·ater, killed and fixed in 70% ethanul. t e (1972) but were referred to by Hanumante (1975). There are The dissections were carried out dorsally using tungsten nee­ n i also contradictions concerning the relative position of these dles and studied under a Wild Heerbrugg M5A dissecting b a organs in the earthworm body. mlcroscope. S

y The morphology of the male reproductive structures has That part of the body containing the reproductive organs b been described (Reinecke & Pieters 1997) and the present was used for sectioning for light and electron microscopy. For d e c u d o r p e R S. Afr. 1. ZooL 1998,33(3) 167

light microscopy the worms were rinsed in distilled water and and 9 contain one pair of spermathecae and one pair of sper­ the anterior third of the body, containing the reproductive mathecal pores. organs, was fixed in aqueous Bouin's for 3 h (Humason 1979). For embedding in paraffin wax, samples were dehy­ Morphology of the female reproductive system drated in a graded ethanol series (50%; 70%; 90%; 100%) For the sake of simplicity, the female organs and structures and placed into a I: I ethanol:chloroform mixture before are described from anterior to posterior. transferring to 100% chloroform. Tissues were exposed to three consecutive paraffin wax baths of which the last one Ovaries was exposed to a vacuum of 150 em Hg, before embedding in Histosec. Longitudinal and transverse sections of 5 ).1m thick­ The ovaries of P. eXcCNatus occur in segment 13, which is ness were cut on a '820' Spencer microtome and floated onto one segment removed from the last set of testes (in segment slides pre-treated with poly-L-Iysine (Huang, Gibson, Facer, 11). This is the situation for earthworm families in general Gu & Polak 1983). (Jamieson 1977; Stephenson 1930). The ovaries of P. excava­ Ius are attached ventrally to both sides of the alimentary Sections were stained with Mallory's trichrome stain canal, and their narrower ends are directed towards the ante­ (Humason 1979). The prepared slides were studied under a rior septum of segment 13. Each ovary branches into numer­ Leitz LS Laborlux S high magnification microscope and pho­ ous stalks with mature oocytes. This is in fine with the tomicrographs were taken by a Wild photographic system findings of Hanumante (1975) and Stephenson (1930). using FP4 film. According to Hesse, as quoted by Stephenson (1930), the Material for SEM and TEM was fixed in Todd's fixative following three regions can be distinguished in earthworm for 2 h (Todd 1986) and washed in cacodylate buffer (0.05M; ovaries, viz. (I) the primordial cells are limited to the area of pH ~ 7.4) twice for 10 min each, postfixed in 1% (w/v) aque­ the septum attachment, (2) posterior to the latter is a cell divi­ ous osmium tetroxide for I h (Bullock 1984). The samples sion region, and (3) a region which contains mature oocytes were washed in distilled water after which they were stained that lies posteriorly to the former one. These regions were with 2% uranyl acetate (Tiedt, Jooste & Hamilton-Attwell also observed for Fredericia, LumbricilLus, Tubi/ex and Lim­ 1987). The material was washed in distilled water and dehy­ nodrilus (Stephenson 1930). Hanumante (1975) described the drated in an acetone series (50%; 75%; 95%; 100%; 100%) free ends of the ovaries as lobes containing oocytes of differ­ for 20 min each. ent developing stages in a linear order. During the present his­ For TEM studies the samples were placed in a I: I mixture tological studies the linear rows could not be distinguished. It of acetone and Spurr resin after dehydration (Spurr 1969), was, however, possible to recognise that the oogonial cells transferred to 100% resin and embedded in fresh Spurr's . occur nearest to the attachment and that the more mature )

9 resin. A Reichert Ultracut E ultra microtome was used to cut stages occur further away. 0

0 sections of 100-130 nm thickness which were stained with Stephenson (1930) reported ovary sacs for some of the 2 5% uranyl acetate and lead citrate (Reynolds 1963) and stud­ d perichaete Megascolecidae, but no ovary sacs have been e t ied in a Philips eM 10 transmission electron microscope at observed in subsequent studies on P. excavalus (Gates 1972; a d 100 kY. Hanumante 1975). (

r Material was also prepared for SEM to observe the external e h reproductive organs by critical point drying in liquid carbon The ovum/unneLs, oviducts and/emale apertures s i l dioxide, and after dehydration, mounting on stubs and coating b The ovum funnels of P. excavatus are in the same segment as u with carbon. These samples were studied under a Cambridge

P the ovaries and this corresponds with the general position for

e Stereoscan 250 SEM. ovum funnels of earthworms (Stephenson 1930). They are h t attached ventrally to the posterior septum of segment 13. y b Results and discussion

Hanumante (1975) described them as saucer-shaped with cili­ d

e ated edges. The ovum funnels are revealed by histological

t Number and position of female reproductive structures n studies only (Figure I B). a Reinecke & Pieters (1997) diagrammatically represented the r TEM micrographs showed that the ovum funnels of P. g positions of all the reproductive structures (Refer: Figure I A e excavatus consist of a single layer of ciliated columnar epi­ c in Reinecke & Pieters 1997). One pair of ovaries and one pair n thelium. These cells are arranged with their ciliated ends e c of ovum funnels connected to one pair of short oviducts are i pointing to the opening of the funnel and their nuclei are l

r found in segment 13. This confirms the findings of Hanu­ basally situated (Figure I C). The cytoplasm of these cells e d mante (1975). In segment 14 the oviducts join to form a short clearly reveals endoplasmic reticula and mitochondria. A n u common oviduct, ending in the single female reproductive large number of Golgi complexes are present, as well as

y opening. The latter is situated in the centre of the segment,

a secretory vesicles (Figure I D). An amorphous substance is w ventral and directly anterior to the ring of body chaetae (Fig­ also observed between the cilia. e t ure I A). The position of the female aperture observed in this a The oviducts, originating in the funnels, pass through the

G study supports the findings of Beddard (1886), and there is posterior septum of segment 13 and unite as a common ovi­ t e thus agreement that the female aperture of P. excavatus is on duct that opens midventrally on segment 14. The position of n i segment 14. All the worms investigated in the present study

b the opening coincides with what Stephenson (1930) a had a single, central female aperture, however Beddard described. Although the ovaries are posterior to the testes, the S

y (1886) reported the female aperture in different segments, and female opening is anterior to the male ones. b

d claimed that there were more than one. Each of segments 7, 8 The oviducts of P. excavalus consist of a single layer of e c u d o r p e R 168 S. Afr. J. Zoo I. 1998.33(3) . ) 9 0 0 2 d e t a d ( r e h s i l b u

P -,

e Figure 1 (A) TEM micrograph of ventral view of segment 14 , show in g the position of the female reproductive opening (FO), (BC =:; body clul· h t

elae); bar = 1.0 Ilm. (8) Light micrograph of longitud inal secti on through segmen t 14 showing an ovum funnel (Or); bar = 10.0 11m . (C) TEM y b micrograph of an ovum funnel displaying the ciliated co lumnar epi thelial cells (eE) with nuclei (N), (C = I.:ilia); bar = 10.0 j..lm . (D) TEM

d = Ilm. e micrograph depicting vesicles (V) and cilia (e) in the ovum funnel ; bar 1.0 t n a r g

e ciliated epithelium and this observation confirm s that of latter finding is supported by the present study. c n Stephenson (1930). The epithelial layer is surrounded by con­ e Our findings as to the relative position of the spcrmathecal c i nective tissue. Muscle fibres are present in close proximity to l pores agree with those of Beddard (1886) and Gates (1972)

r the connective tissue (Figure 2A). These muscle fibres may e who also found them to be s ituated midventrally. We d aid the cilia in tran sporting the ova.

n observed the pores in the intersegmental fold (Figure 20). u

Although the female opening is single (Figure I A), sec­ anterior to the segment in which the spermathecae occur. y a tions showed that it is fonned by the two separate oviducts Hanumante (1975), however. described the openings as vent­ w e which are joined together (Figure 2B). rolateral. The spermathecae open close together but clearly t a separate from each other (Figure 20). The spermathecal pores G Spermalhecae and spermalheca/ pores t seen during this investigation have no cilia, chaetae or surface e

n ornamentation .

i A pair of sp.ermathecae are situated ventrally and on both b

a sides of the digestive tract (Figure 2C), in each of three seg­ Each opening gives ri se to a small canal, the spe rmathecal S ments. Although Beddard (1886) described only two pairs of duct. This duct widens into a large ampulla, as described by y b

spennathecae Hanumante (1975) mentioned three pairs. The Stephenson ( 1930). At the base of the ampulla there are four d e c u d o r p e R S. Afr. 1. 2001. 1998,33(3) 169 . ) 9 0 0 2 d e t a d ( r e h s i l b u P e h t y b d e t n a r g e c n e c i l r e d n u y a w e t Figure 2 (A) TEM mi crograph of oviduct wall of P. excavalus with lumen (OL) on the len. (CT = connective tissue; M= muscle fibres); bar = a

G 2.0 )lm. (8) I.ight micrograph of longitudinal sect ion through female opening showing ils double nature (1'); bar = 10.0 ~m. (e) Light micro­ t

e graph of cross section through a segment containing one pair of sperm at he cae (S), (DT -"'- digestive tract); bar = 50.0 ~ l m . (D) SEM micrograph n i of ventral side of an intersegmental groove showing the position orlhe spermathecal pores (i); bar =.: I 0.0 ~tm . (E) Light mi crograph or dis­ b a sected P excavalus specimen. showing the relative posi tions of the spermathecal diverticula (D) and spermalhecal ampullae (A). (NC = ven­ S = y tral nerve cord). (The ampullae have been emptied to make the underlying diverticula more visible): bar I 0.0 ~m . (F) Light micrograph of b ampulla (A) and connected diverticula (D) of a spennatheca.. (SZ = spennatozoa); bar = 2.0 ~m. d e c u d o r p e R 170 S. Afr. J. Zool. 1998,33(3)

diverticula (Figure 2E). According to Stephenson (J 930) the confined singly. In hi sto logical sec tion s sperm were also prese nce of these diverticula is common for most mem bers of observed in th e bases of the ampullae of mated worms (Fig­ the Megascolecidae, as well as many Enchytraeidae and a few ure 2F). Lumbricidae. Some diverticu la may be simple sac-like evagi­ The wall s of th e spermatheca l ducts consist of a stratified nations attached directly to the base of the ampulla or th ey epithelium surrounded by connective ti ssue which is in agree­ may be connected by a slender stal k. The di verticula of P. ment with Jamieson 's ( 1981 ) findings. The ce ll layer border­ excavalus are without stalks (Figure 2F). Dissections of ing on the lumen has many mic rovill i (Figure 3A). A layer of mated specimen s of P. excavatus reveal these diverticula as amorphou s substance is seen to occur between the microvilli . white, glossy structures, clearly distinguishable from the dull There are no secretory granu les visible in the cells comp ri sing surface of the ampullae. the duct wall, but many vesicles are seen, and the membrane s The iridescen t character is attributed to the presence of of some of them fuse with the apical cell membrane (Figure spenn exchanged by mating, and was only present in wonns 38). In contrast, epithelial cells of th e spermathecal duct of that were cultured in batche s. It was never observed in worms Tubi/ex lubi/ex have distinct secretory granules (Jamieson . ) 9 0 0 2 d e t a d ( r e h s i l b u P e h t y b d e t n a r g e c n e c i l r e d n u y a w e t a G t e Figure 3 (A) TEM micrograph of long itudinal section through a spermalheca! duct; bar = 10.0 11m. (8) TEM micrograph of the spermathecal n i

b duci. shown in CA) at higher magnification, showing villi and secretion in the lumen; bar = 1.0 11m. (C) Light micrograph of spennathecal a = ).lm . S diverticulum showin g the slit-like spaces (i) filled with spennatozoa; bar 10.0 (D) TEM mi crograph of the contents of a sperm athecal

y diverticulum; bar = 2.0 ).lm. CBl = basal lamina., G = granules, GC = granular ce ll, L = spermathecal duct lum en. M = mu scle tissue. MV = b

d microvilli. SC = secretion, S2 = spermat ozoa, V = vescides .) e c u d o r p e R S. Mc. J. Zoo I. 1998,33(3) 171

1981). Surrounding the basal lamina of the spemlathecal duct microscop:y': a rcvie\".. J .. \/icr 133: 1 15. is a circular muscle layer probably responsible for the CHRISTENSEN, \3, 1980. Animall..:ytogcllctics. Yol.2. Annt.:lida. entrance and exit of the mating partner's sperm into and out GebrOuer BDrntracg,cr. Bcrlin. JRpp. of the ,pennatheca (Figure 3A). This muscle layer stretches EOWARDS, c.:\. & LOFTY. J .R. 1972. l3ioll)g:y of t.:arthwonns. from the spermathecal pore up to the ventral base of the Chapman and I-Iall. London. 332pp GAlES. U.E. 1972. Burmese earthworms hans, Am. Phil. Soc. ampulla. 62(7): 138-148. Each diverticulum is spherically shaped and consists HALLAT!' L.. REINECKE. A..!. & VILJOEN. SA 1990. Lik mainly of secretory cells. It has a fine canal system inter­ cyclc of the oriental compost worm Pe/"lOny.t excavallls spaced with atria (Figure 3C). Each atrium is filled with an (Ohgochaeta). SAfe.!Zoo/. 25( I): 41-45. amorphous substance of unknown origin, which may be a IlALLAT!' L.. VIl..IOEl\, SA. & REINECKE. A..!. 1<)<)2. Moisture secretory product of the cells (Figure 3 D). According to rcquircments in the life cycle of Perionyx eXC(lvatus Stephenson (1930) the secretion has a nutritious function, (Oligochaeta). SoliBio/. Biochem. 24( 12). 1333-1340. since sperm are stored there until cocoon formation com­ HANUMANTE. M.M. 1975. On the anatomy ofthc reproductive mences. Varute & More (1971) found that the mucous sub­ system of the earthworm: PCriOIl:VX cxcavaws. Alarathwada Universi(vJ .)'ci, (}3io!. Sci.) 15(8): 193-197. stance secreted by the glandular mucosa of the spermathecae HCANG. WM, GIBSOl\. S..I .. FACER. P. GU ..t. & POLAK . .1.M. of Pheretima elongata and Jlap/ochaele//a powe/fi contain 1983. Improved section adbe"ion for immunocytochemistry using glycogen and carboxyl-containing mucopolysaccharides, the high molecular 'weight polymers or Iyslnc as a slide coatmg former probably functioning as energy reserve for stored HistochemIstry 77: 275-279. spermatozoa and the latter in preparing the necessary chemi­ HCMASON, (i.L 1979. Animalllsslie techniqucs. W .l-I. Fr~t!mal1 cal environment for sperm storage. and Company, San Francisco. 661 pro The wall of the ampulla is lined with a stratifted epithelium JAMIESON, RG,M. 1977. On thc phylogeny of tile and consists of granular cells, which according to Stephenson Moniligastridac, with description of a ncw species of MOl1ilig(/ster (1930) have a secreting function. It seems that the mucus con­ (Oligochacta, Annelida). I':vol theory 2: 95-114. taining cd!:.. are of the apocrine secretory type. The ampulla is JAMIESON, RG.M. 1981. The ultrastructure or the Oligochaeta. surrounded by connective tissue. The function of the ampulla, A<:ademic Press, London. 462pp. NEUHACSER. E.F .. I.OEHR. R.C. & MAI.ECKI. M.R. 1<)88 The other than for additional storage of sperm, is unknown in p, potential of earthworms for managing s~wagc sludge. In: excavatus. According to Richards & Fleming (1982) the epi­ Earth\vonns in \vaste and environmentallllanagemt:nt.(Eds) thelium of spermathecae of Dendrohaena suhruhicunda, Edw'ards, C.A & Ncuhauser. Lr, SPB Academic Puhlishing. The three A//%/Jophora species and Lumbricus rubel/us phago­ Hague. pp. 9-20. cytoses the sperm, but no evidence of this could be found in REINECKE, AJ. & IIALLAIT, L. 1989. Growth and cocoon .

) P. excavatus, production or Pa/onyx excamlus (Oltgocllada). Bioi. Fcl'/ ..\oils 9

0 8: 303-306. 0

2 REINECKE. SA & PIETERS. R 1997 The morphology oflhe

Conclusion d malc reproductive structures of Perioll.n excavatlls (Oligocilaeta). e t During this study, several aspects concerning the female S ;lfr J Zool. 32(3): 64 71. a d reproductive system of P. excavatus were elucidated. How­ REINECKE, AJ. & REINECKE. S.A. 191)4, Influcnce ofv,'onn (

r ever, after investigating the female structures in this study, as density on growth and cocoon productIon or thc Asiatic e

h well as the male organs (Reinecke & Pieters 1997), no mor­ earthworm Perionyx excavalus (Oligodmeta. Megasculecidae). s i l phological grounds could be found for self-fertilization. E",. J Soil Riol. 30( I) 2933. b REYNOLDS. E.S. 1963. The use Df kad citrate at high pll as an u The possibility that spenn cells could migrate through the

P (.t'l{ BiOI. 17: septa into adjacent ova-containing segments and fertilise electron Dpaque stain in electron microscopy. J. e 208-213. h

t these ova before being deposited into cocoons cannot be ruled REYNOLDS. J.W. 1974. Are oligocilactt:s really hermaphroditic y out. According to Stephenson ( 1930), the migration of sperm b amphimictic organisms? Bi()/ogfsf 56(2): 90-'J9.

d through the septa occurs in many members of the Eudrilidae. & 'II'

e RICHARDS. K.S. FLEMING. 1982. Spermatozoal t During the present study, however, no spenn cells were found phagocytosis hy the :::.permathecac /)l!J!drobacna subrllbiclinda n or a in the diverticula of worms raised singly. The only otherpos­ and Dther lumbricids (Oligochacta. Alll1dida). J lllvl!l'lebr r Int. g sible explanation f()f the fact that single individuals of this lIeprod. 5: 233-241. e c species form viable cocoons without having any contact with SPCRR. AR. I 96(). A low-vi:.cosity epO\.y rt:sin embedding n e uther individuals during their lifetime, could be parthenogen­ medium for electron microscopy_.1. l//traslruc, Res, 26: 3 I -43. c i l STEPHENSON. J. 1930. Thc Oligochaeta. Whc1doll & Weslc~. esis. This has been found tor Pherelima-species, some Tubifi­ r e cidae, Enchytraeidae and Lumbricidae (Christensen 1980; New York, 978pp. d TIEDT, L.R .. .Ioosn:. W..1. & HAMIL lON-AITWELL. VL n Reynolds 1974) and needs tu be studied further in P. excava­ u 1987. Technique for preserving at:rial fungus structures for l1IS. y scanning c1ectron microscopy. trails IJrrtish .'1{vL'. Soc 88(3)' a

w 420-422. e t References rOOD, W..!. 1986. EJll'cts of specimen prepnration Dn the apparent a BEDDARD. F.E. 1886. Descriptions of some new Dr litlk-know'n ultrastructure ofmicrourganisms, In. Ultrastructure techniques for G

t earthworm::. together with an ac(;uunt of the variatiuns in structure microorganisms. (Eds) Aldrich, I I.e. & Todd. V./.J. Plenum Pn::.s. e n exhibited by Perionyx excavalus. Proc. Zoo/. Soc Lond. 21: New' York. pp. 87-100. i b 2Y8-314. VARUTE. A.T. & MORE. N.K. 1971. C) (oehemieal >tody of a

S BEDDARD. f.E, 1892. On some new species from various parts of mucus and mucus-secreting cells in spermo.thcclic of the

y tbc world. Proc. /.001. Soc I.ond. 46: 684---69(), earthworms, Pherelinw eiongaJa (Perrier) and lIoplochacteila b BUI.I.OCK, G.R. 1984. The current status of fixation for electron powelli (Michaelsen). India/l J F,p. B/()1. 10: 239-24 I. d e c u d o r p e R