parasite Cichlidogyrus haili typicus has a short oviduct/ova-vitelline duct separated from the ... halli typicus; monogenean; gill parasite; oviduct; ovo-vitelline duct;.
ULTRASTRUCTURAL OBSERVATIONS ON THE OVIDUCT, MEHLIS’ GLANDS AND OOTYPE OF THE MONOGENEAN CICHLlDOGYRUS HALLI TYPICUS (PRICE & KIRK, 1967) PAPERNA, 1979 M. M. EL-NAGCAR,*
A. A. KHIDR~ and G. C. KEARN$$
* Zoology Department, Faculty of Sciences, Mansoura University, Mansoura, Egypt t Zoology Department, Faculty of Sciences, Mansoura University, Demietta Branch, Demietta, Egypt $ School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K. (Received 20 July 1989; accepted 12 October 1989) A~s~Fs~--EL-NAGGAR M. M., KHIDRA. A. and KEARNG. C. 1990. Ultrast~ctuml observations on the oviduct, Mehhs’ glands and ootype of the monogenean Cichlidogyrus ha& typieus (Price & Kirk, 1967) Paperna, 1979. Znternational Journalfor Purmitolugy 20: 203-209. The ancyrocephaline monogenean gill parasite Cichlidogyrus haili typicus has a short oviduct/ova-vitelline duct separated from the germarium and from the ootype by sphincter muscles. The chamber so-formed contains spermatozoa, the axonemal ends of which appear to be attached in an intimate way between lamellated extensions of the chamber lining. This arrangement may facilitate fertilization by retaining an oocyte temporarily in close contact with the free nucleated ends of the spermatozoa, i.e. the short oviduct/ova-vitelline duct may serve as a fertilization chamber. There are two distinct types of Mehlis’ gland opening at the base of the ootype and the latter has a cellular, non-secretory lining which is not penetrated by gland ducts.
INDEX KEY WORDS: Cichlidogyrw halli typicus; monogenean; gill parasite; oviduct; ovo-vitelline duct; Mehlis’ glands; ootype; sperm; fertilization.
INTRODUCTION KEARN (1986) has pointed out that the monogeneans
are probably the least specialized of the three major groups of parasitic platyhelminths and may have diverged least of all in terms of ootype architecture and function from their free-living platyhelminth ancestors. In spite of this, there has been only one detailed ultrastructural study of the glands (Mehlis’ glands) associated with the ootype and of the ootype itself in monogeneans, namely that of Stranock & Halton (1975) in which they compared the monocotylid Caiicotyle kmyeri with the polyopisth~otyle~ ~~10~00~ ~a~~do~~~, and there are no extensive comparative studies of the Mehlis’ glands and their deployment similar to those made on cestodes and digeneans. For these reasons, an ultrastructural study of the female reproductive tract of the dactylogyroidean (ancyrocephaline) monogenean Cichlidogyms halli typicus (Price & Kirk, 1967) Paperna, 1979 seemed highly desirable. MATERIALS AND METHODS The freshwater cichlid fishes Sarotherodon (= Tiiapiu) nilotica, S. galilaen and Tifapia ziNii were caught by netting
from the Demietta branch of the River Nile near Mansoura and Demietta, Egypt. The fishes were kept alive until required in tanks containing circulating river-water. Living $ To whom all correspondence should be addressed. 203
parasites from the gills were preserved for 2-3 h in 2.5% glutaraldehyde buffered to pH 7.4 with 0.1 M-sodium cacodylate_HCl containing 3% sucrose and 0.01% calcium chloride, washed in several changes of cold buffer and then post-fixed for 30 min in 1% osmium tetroxide in sodium cacodylate buffer. The specimens were processed either for semi-thin sectioning and staining with 1% toluidine blue in 1% borax or for transmission electron microscopy (TEM) as described by El-Naggar & Kearn (1983). TheTEM work was conducted by the second author (A.K.) at the University of East Anglia, Norwich, U.K. RESULTS The ovidu~~lovo-v~~elli~e duct
system of C~ch~~dogy~~~ in Fig. 1. ElNaggar & Khidr (1985) reported that the germarium and the ootype in C. halli typicus are connected by a short straight duct into which open two transverse vitelline ducts, one from each side. The point of entry of the vitelline ducts has not been precisely determined but it appears to lie close to the ootype. The vagina, which expands to form a receptaculum seminis, also appears to join the oviduct at its proximal end but this communication has not been identified with TEM. No anatomical features were found that could be used to distin~ish between the oviduct and the ovo-vitelline duct in TEM sections. TEM observations have revealed that the lumen of the oviduct/ova-viteIline duct is lined by a single layer The female reproductive
huBi typicus is shown diagrammaticaIly
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M. M. EL-NAGGAR,A. A. KHIDRand C. C. KEARN
of epitheiial cells (Fig. 2). The thickness of the lining epithelium gradually increases from the germarium to the ootype and its iuminal surface is elevated into lamellae, many of which branch or loop to rejoin the surface. A remarkably uniform spacing often exists between the plasma membranes of adjacent iamelfae (Figs. 2, 3). These membranes run parallel for long distances separated by a uniform gap of about 18 nm containing homogeneous, moderately electron-dense material. The lamellae reach their greatest length at the proximal end of the oviduct, near the germarium and this region of the oviduct contains many spermatozoa which often lie between the lamellae. The
adjacent plasma membranes of the spermatozaa and the tamellae enclosing them have an intimate spatial relationship similar to that occurring between the plasma membranes of adjacent lamellae (Figs. 2, 4). Some of the spermatozoa are ensheathed by a wrapping provided by a single lamella and others are enclosed between two adjacent lamellae, but in both of these situations, a precise 18 nm gap exists between the adjacent membranes of the sperm and the lamella, In the situation where the sperm is sandwiched between two lamellae, the adjacent plasma membranes ofthese two lamellae maintain the same 18 nm spacing before and after separating to accommodate the sperm (Fig.
,Gl
FIG. 1. The female reproductive system of C~c~~~~ug~rffs halti typicus (ventral view) reconstructed from light micrrtscope and TEM observations. For convenience, Mehlis’ glands have been omitted from one side of the diagram. co, common genitaf opening; GI, M&is gland producing vesicular bodies; G2, M&is grandproducing =&&on-dense bodies; g, germarium; o, ootype; ov, oviduct/ova-vitelline duct containing attached spermatozoa; rs, receptaculum seminis eantaiaing spermatozoa; sm, sphincter muscle; vd, vitelline duet; ~5, vaginal opening. Scale bar = 25 ,mn.
Female reproductive system of Cjc~f~~~y~s
FIG. 2. Schematic drawing of a TEM section through one ofthe cells lining the oviduct of ~jc~Z~~og~r~huZ(itypicus.Note the intimate relationship between the membranes of adjacent lamelfae (1) and between the membranes of lamellae and spermatozoa(s). a, axoneme; bl, basal lamina; bm, basal plasma membrane; c, junctional complex; ger, granularendoplasmic reticulum; go, Go&i body; lu, lumen of oviduct; m, mitochondrion; n, nucleus; r, ribosomes; ser, smooth endoplasmic reticulum. Scale bar = 0.5 Brn.
of C. halli typicus has been reported by Khidr (unpublished PhD thesis, University of Mansoura, Egypt, 1986) to consist of a single axoneme, a nucleus and a mitochondrion. At one end the axoneme projects beyond the nucleus and the mitochondrion whereas at the other end the sperm contains only the nucleus and a few microtubules. In the present study it was noted that transverse sections of many of the s~~atozoa enclosed between the lamellate extensions of the epithelial cells contained only the axoneme (Figs. 2,3,4). Each cell of the lining epithelium of the oviduct possesses a single irregularly-shaped nucleus and its cytoplasm contains mitochondria, granular endoplasmic reticulum (GER), smooth endoplasmic reticulum, Golgi bodies and numerous ribosomes (Fig. 2). Beneath the basal lamina lies a continuous layer of circular muscle fibres and outside this layer there are isolated groups of longitudinal muscle fibres. Semi-thin sections stained with toluidine blue have revealed that the opening of the germarium into the oviduct and the opening of the ova-vitelli~e duct into the ootype are each surrounded by a prominent sphincter of muscle fibres. This has been confirmed with TEM (Fig. 5). 4). The mature spermatozoon
Mehlis ’ glands
Using TEM, two kinds of Mehlis’ gland have been identified. One of these glands (Fig. 1, Gl) contains distinctive spherical secretory bodies with vesicular contents and the other (Fig. 1, G2) contains spherical or ovoid electron-dense bodies. The latter cells were identified using light microscopy by El-Naggar B Khidr (1985). The ducts from all of these cells open close together into the female repr~uctive tract at the junction of the ovo-vitelline duct and the ootype but the ducts carrying the vesicular bodies open distal to those carrying the electron-dense bodies, i.e. the ducts carrying the vesicular bodies open closer to the ootype (Figs. 1, 5). Where it penetrates the ootype entrance, each Gl and G2 duct is lined by microtubules. The cells producing the vesicular bodies are more abundant than the other gland cells and are located ventrally, lateral to the posterior half of the ge~arium (Fig. 1). Much of their cytoplasm is packed with secretion but Golgi bodies and narrow cisternae of GER are also present, although the latter is mostly restricted to the perinuclear and peripheral regions of the cell (Fig. 6). The membrane-bound secretory bodies contain vesicles of two types, namely small ovoid or etongate vesicles each with a dense boundary
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and large vesicles with moderately electron-dense contents including occasional electron-lucent areas or electron-dense particles. The cells producing the electron~en~ bodies are also located ventrally but they are more anteriorly situated lateral to the oviduct (Fig. 1). Their cytoplasm contains extensive, characteristically dilated cisternae of GER, elongated mitochondria and Golgi bodies (Fig. 7). In addition to the mature secretory bodies which are spherical or ovoid, membrane-bound, homogeneous and electron-dense, there are larger bodies with fibrous or lamellate contents. The ootype The lumen of the ootype is lined by a single layer of uninucleated epithelial cells (Fig. 8). Adjacent ceils are joined by junctional complexes. The luminai surface of these cells is elevated to produce lameliae and in many places, where two lameliae come into contact with each other, the adjacent plasma membranes are separated by a uniform 18 nm gap as in the oviduct. The gap contains an electron-dense core embedded in an electron-lucent ground substance. Spermatozoa were frequently encountered in the ootype lumen and some of these were found to be enveloped by the ootype Iamellae in a manner similar to that observed in the oviduct. The cytoplasm of the luminal epithelial ceils contains a moderate amount of GER, numerous ribosomes and a few Golgi bodies. There is no evidence that these epithehal cells produce secretory bodies and no gland ducts were found penetrating the wall of the ootype. Beneath the basal lamina, two layers of muscle fibres are present, an inner circular and an outer longitudinal layer. At the junction of the ootype and the ovo-vitelline duct, the sphincter muscle cells contain mitochondria and glycogen-like particles in their sarcoplasm. Remnants of vesicular secretory bodies (see below) were found in the ootype lumen.
DISCUSSiON
An account has been given of the ultrastructure of the oviduct, Mehlis’ glands and ootype of the ancyrocephaIine gill parasite ~ic~f~~~~~rus halfi typicus (Price & Kirk, 1967) Paperna, 1979. It has emerged that the ovidu~t/ovo-~teliine duct serves not only for transport of the oocyte (zygote?) to the ootype but also as a sperm store. The most significant discovery is that there is an intimate association between the spermatozoa and the lamellate extensions of the cells lining the female reproductive tract, especially at the proximal end of the oviduct. The plasma membranes covering the spermatozoa and the lamellae are in close apposition, with a uniform 18 nm gap between them. The uniformity of this close spacing argues against a casual juxtaposition and suggests that the adjacent membranes may be intimately bound together in a more ~~anent way. A few spermatozoa were found to be associated in a similar way with the lamellar extensions at the proximal end of the ootype. The relationship is reminiscent of the situation in Schistosoma mansoni reported by Erasmus (1973) who suggested that a transfer of nutrients may take place between the oviduct lining and the spermatozoa. In the nematode ~ipetaIo~ema viteae, Foor, Johnson & Beaver (1971) described a similar phenomenon and suggested that sperm retention was necessary for maturation. According to Khidr (1984, thesis cited above) the two ends of the filiform sperm of C. halii typicus are different. The single axoneme, but not the elongated nucleus and mitochondrion, extends to one end of the sperm while at the opposite end the nucleus extends beyond the mitochondrion and only a few of the axonemal microtubules persist alongside the nucleus. The evidence in the present paper suggests that it is the axonemal end of the sperm that is associated with the
Fro. 3. TEM section through the proximal region of the oviduct showing the intimate relationship (small opposing arrowheads) between spermatozoa and lamellar extensions of the duct lining. A similar relationship can be seen between adjacent lamellae (large opposing arrow-heads). Scale bar = I pm.
Fro. 4. Part of Fig. 3 at higher ma~ification.
I, lamella; s, s~~atozoon.
Scale bar = 0.25 pm.
FIG. 5. TEM section through the junction between the ovo-vitelline duct (ov) and the ootype (0). Sl, S2, vesicular and electrondense secretory bodies, respectively. These bodies lie within their ducts which have been sectioned close to the point where they enter the lumen at the proximal end of the ootype. The excessively large amount of sperm in the lumen of the ovo-vitelline duct and the ootype is atypical; the sperm may have been displaced from a more proximal location by contraction at the time of preservation. n, nucleus of ceil lining the ovo-vitelline duct; sm, sphincter muscle. ScaIe bar = 2 pm. FIG. 6. TEM section of a gland cell producing the vesicular secretion (SI). ger, granular endoplasmic reticulum; n, nucleus. Scale bar = 1 pm. FIG. 7. TEM section of a gland cell producing the eiectron-dense secretion (S2). f, fibrous or lamellate precursors of S2 bodies; ger, granular endoplasmic sticky; n, nucleus. Scale bar = 0.5 pm.
FIGS.3-7.
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M. M. EL-NAGGAR,
A. A. KHIDR and
G. C. KEARN
FIG. 8. TEM section through the epithelial lining of the ootype. ger, granular endoplasmic reticulum; i, infoldings of basal lamina and basement membrane; 1,lamella; lu, lumen of ootype; m, mitochondrion; mu, muscle; n, nucleus; s, spermatozoon. Scale bar = 1 Grn.
oviducal lamellae, with the nuclear end of the sperm lying free in the lumen. If the bond between the sperm and the lamellae is sufficiently strong, then this link and the orientation of the sperm might have important advantages. The link with the lamellae would prevent the drifting of spermatozoa in a distal direction as a result of the entry of an oocyte from the germarium and peristaltic movements of the oviduct wall, and the orientation of the sperm would favour entry of the sperm nucleus into the oocyte. Thus, it is possible that the oviduct of C. halli typicus acts as a fertilization chamber where the oocyte is held temporarily in close contact with spermatozoa before passing on into the ootype. Compatible with this function is the presence of isolating sphincter muscles at the proximal and distal ends of the chamber and also the relatively distal point of entry of the vitelline ducts. Calicotyle kroyeri is the only other ‘monopisthocotylean’ monogenean in which the reproductive tract has been studied with the TEM (see Stranock & Halton, 1975). Stranock & Halton made no reference to glands opening at the ootype entrance and reported a secretory, cellular ootype lining penetrated by ducts from two distinct kinds of Mehlis’ gland cells. In contrast, C. halli typicus has two distinct kinds of gland with ducts leading to the ootype entrance and a cellular, but apparently non-secretory, ootype epithelium which is not perforated by ducts from external glands. Thus, C. halli typicus conforms with the distinction based on light microscopy made by Goto (1894) between ‘monopisthocotyleans’ with
ootype entrance glands only and ‘polyopisthocotyleans’ which were said to possess additional glands opening through the ootype wall. Cerfontaine (1898) described two kinds of ootype entrance glands in the monocotylid Merizocotyle diaphanum but he considered that the two kinds may be developmental stages of the same gland. Kearn (1978) also reported two kinds of ootype entrance glands in Entobdella australis but he suggested that one of these secretions might be concerned with attachment of the eggs to the substrate. It is not likely that the two secretions of C. halli typicus represent developmental stages of the same secretion and there is no evidence that the eggs carry adhesive material. The large bodies containing fibrous or lamellate material in the cytoplasm of the gland cell producing the electrondense secretion seem likely to be precursors of the electron-dense secretory bodies. The two Mehlis’ secretions of C. halli typicus have ultrastructural features which correspond with those of the two Mehlis’ secretions of the digenean Quinqueserialis quinqueserialis as described by Holy & Wittrock (1986). The vesicular nature of the Gl secretory bodies in C. halli typicus is reminiscent of the so-called alpha secretion of Q. quinqueserialis and the electron-dense G2 bodies resemble the beta secretory bodies of the digenean. These similarities between the rather distantly related monogeneans and digeneans suggest that the glandular apparatus involved in the assembly of ‘tanned’ eggs may be basically the same throughout the parasitic platyhelminths. However,
Female reproductive system of Cichlidogyrus
further speculation on homologies and functions of glands associated with the ootype in monogeneans seems inapprop~ate at this time because of the limitations
of our knowledge.
AcknowledgementsWe
are grateful to Mr B. C. Bird for advice on electron microscopy. REFERENCES CERFONTAINE P. 1898. Le genre Merizocotyle (Cerf). Archives de Biologie 15: 329-366. EL-NAGGARM. M. & KEARNG. C. 1983. Glands associated
with the anterior adhesive areas and body margins in the skin parasitic monogenean Entobdeila soleae. International Journaljbr Parasitology 13: 67-81. EL-NAGGARM. M. & KHIDR A. A. 1985. Redescription of the monogenean gill parasite Cichl~dogyrus halli iypicus (Price & Kirk, 1967) Papema, 1979, from Tilapia spp. in Egypt. First International Conference of AppIied Sciences, Zagazig University 4: 138-l 60. ERASMUS D. A. 1973. A comparative study of the reproductive system of mature, immature and ‘unisexual’ female Schistosoma mansoni. Parasitology 67: 175-183. FOOR W.
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Mo~hologic~ changes in the spermatozoa of Dipetalonema viieae in utero. JournalofPar~ifology 57: 1163-I 169. GOTO S. 1894. Studies on the ectoparasitic trematodes of Japan. Journai ofthe College ofScience, Imperial University of Tokyo 8: l-273. H&LV J. M. & WITTROCK D. D. 1986. Ultrastructure of the female reproductive organs (ovary, vitellaria, and Mehlis’ gland) of Hahpegus eccentricus (Trematoda: Derogenidae). Canadian Journal of Zoology 64: 2203-2212. _ KEARN G. C. 1978. Entobdella australis, sp. nov., a skin
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