ORIGINAL PAPER

Light and electron microscopic study on Henneguyasuprabranchiae Landsberg, 1987 (Myxozoa: Myxosporea)infecting Oreochromis niloticus, a new host record

Fathy Abdel-Ghaffar & Abdel-Azeem Sh. Abdel-Baki &Elsayed M. Bayoumy & Abdel-Rahman Bashtar &

Saleh Al Qurieshy & Kareem S. Morsey & Ali Alghamdy &

Heinz Mehlhorn

Received: 10 April 2008 /Accepted: 22 April 2008 / Published online: 1 June 2008# Springer-Verlag 2008

Abstract Out of 58 live tilapia fish, five Oreochromisniloticus were found to be naturally infected with Henneguyasuprabranchiae (8.62%). Such infection was recorded onlyduring winter season from Bahr Shebin, a tributary of theRiver Nile at Menoufia Governorate, Nile Delta, Egypt.Based on the structure and measurements of fresh spores,this parasite was identified as H. suprabranchiae. Spores areoval in shape and they measure 15 (13–16)×5 (4–6) µmlength by width. It has two polar capsules inside and theymeasure 4 (5–7)×1 (2–3) μm length by width. Each polarcapsule has spirally coiled (7–9 turns) polar filament. Theplasmodia as well as all other parasitic stages were describedusing light and transmission electron microscopy and

discussed regarding to those of other fish hosts especiallythose of Africa.

Introduction

Myxosporea Buetschli, 1881 are mainly fish parasites. Theyform an abundant and diversified group of parasites. Morethan 100 species of them are known to infect freshwater,brackish, and marine fishes in Africa (Fomena and Bouix1997) and this number is continuously increasing. Due totheir economic importance, myxosporean parasites infect-ing Egyptian fishes have received much attention during thelast decade (Abdel-Ghaffar et al. 1995, 2005; Ali 1999;El-Mansy 2002). Some of the recorded species were provento cause adverse effects on the fish population as causativeagents of severe diseases to fresh and marine water fishes(Kostoingue et al. 2001; Kent et al. 2001; Adriano et al.2005; Cuadrado et al. 2007). The immune response of thehost against these parasites is manifested in severalreactions. Inflammation is usually the principal defensemechanism. It sets in typically at a time when thetrophozoite is replete with mature spores (Klontz et al.1986; Dykova and Lom 1987) or when the parasitedevelops in an atypical site (Komourdijia et al. 1977;Grupcheva at al. 1985; Lom and Dykova 1992) orabnormal host (Seagrave et al. 1980). Investigation byelectron microscopy is not only of major importance tounderstand the myxosporean complicated life cycle andbiology but is also a valuable taxonomic tool and providesinformation aiding in understanding features that maycontribute to pathogenicity (Abdel-Ghaffar et al. 1995,2005). The present study was aimed to study the Henneguya

Parasitol Res (2008) 103:609–617DOI 10.1007/s00436-008-1019-z

F. Abdel-Ghaffar (*) :A.-R. Bashtar :K. S. MorseyZoology Department, Faulty of Science, Cairo University,Giza, Egypte-mail: fathyghaffar@yahoo.com

A.-A. S. Abdel-BakiZoology Department, Faculty of Science, Beni-Suef University,Beni-Suef, Egypt

E. M. BayoumyHydrobiology Department, National Research Center,Giza, Egypt

S. Al Qurieshy :A. AlghamdyZoology Department, College of Science, King Saud University,Riyadh, Saudi Arabia

H. MehlhornZoomorphology, Cell Biology and Parasitology Institute,Düsseldorf University,Düsseldorf, Germany

species recorded in the new host Oreochromis niloticus andto shed light on its classification.

Materials and methods

A total of 58 live Oreochromis niloticus were randomlycollected from Bahr Shebin, a tributary of the River Nile atMenoufia Governorate, Nile Delta, Egypt. Fishes weredirectly transported to the laboratory, using special boxeswith good aeration and cooling, and were kept alive tillexamination for myxosporean infections. The gills wereremoved and placed in Petri dishes with 0.65% salinesolution for microscopic examination. For light microscopy,gill fragments were compressed between glass slides andcover slips for smear preparations. Smears were air-dried atroom temperature, fixed by immersion in absolute methylalcohol, and stained by 1:9 Giemsa/buffer solution for10 min according to Martins and Onaka (2006). Descrip-tion, measurements, and identification of the parasite wereperformed from fresh smears of mature spores using ZeissAxiovert 135 calibrated ocular micrometer (Lom and Noble1984; Lom and Arthur 1989; Martins et al. 1999).

For electron microscopic studies, plasmodia of differentdegrees of maturity were fixed in 2.5% glutaraldehyde in0.1 cacodylate buffer solution (pH 7.4) for 4 h and postfixed in 2% osmium tetroxide for 2 h at 4°C. Specimenswere rinsed twice in the same buffer for 10 min beforedehydrated in ascending series of concentrations of ethanoland finally embedded in Epon araldite according to Luft(1961). Semithin and ultrathin sections were prepared usinga Reichert–Jung ultratome, double stained with uranyl acetateand lead acetate, then examined and photographed by meansof a Philips 400 transmission electron microscope.

Results

Light microscopy

Plasmodia

The plasmodia of this parasite were detected in the gillfilaments. They appeared spherical, whitish in color, andmeasured about 0.77 mm. They were polysporic, contain-ing all sporogenic stages of the parasite (Fig. 1).

Spore description

Mature spores are ovoid in shape in valvular view andappear typically fusiform in lateral view (Figs. 2 and 3).The shell valves are nearly symmetrical and project in twothin and equal caudal processes extending behind the spore.

The spores are 15 (13–16) μm long, 5 (4–6) μm wide, andthe caudal processes are 31 (29–33) μm in length. Thepolar capsules are banana-shaped and taper to their anteriorend. They occupy nearly half of the spore and measure 5.5(5–7) μm in length and 2 (1–3) μm in width. The polarfilament spires in 10–11 coils (Figs. 4 and 5). Thesporoplasms are binucleated, each with a central iodinophilicvacuole.

Electron microscopy

Plasmodia

Transmission electron microscopy revealed that the plas-modia were bordered by a single cell membrane. Theplasmodial wall runs fairly along with the host tissue and isseparated by a narrow gap of electron-lucent materialcovered by a fine granular coat. This wall consists ofdouble unit membrane of high electron density, separatedby a layer of lower electron density. The inner membraneinvaginates into numerous small canals that pinch offpinocytotic vesicles (Fig. 2). Adjacent to the plasmodialwall, the generative cells and the early stages of sporogonyare located peripherally or centrally.

Sporogenesis

It can be clearly seen that the first recognizable stage ofsporogenesis is represented by generative cells. These cellsare ovoid in shape and contain a large nucleus with anucleolus. The homogenous cytoplasm of these cells andother developmental stages contains a number of mito-chondria (Figs. 6 and 7). The development of a generativecell (the enveloping cell) into another stage (the sporont)results in the pansporoblast stage (Figs. 8 and 9).Subsequent development and cell divisions of the sporontinside the enveloping cell occur and the process proceedstowards maturation (Figs. 9, 10, 11, 12, 13, 14, 15, 16, and17). At late sporogenesis, cell differentiation as well as thearrangement of the differentiated cells is seen (Figs. 10, 11,12, 13, 14, and 15). The progeny cells resulting from earlysporogenesis achieved differentiation into capsulogenic,valvogenic, and sporoplasmic cells. Each spore-producingunit consisted of two capsulogenic cells and a sporoplasmsurrounded by two valvogenic cells (Figs. 11, 12, 13, 14,and 15). Maturity of spores exhibited a structural progresstowards capsulogenesis.

Capsulogenesis

An early stage of capsulogenesis is the capsular primordium,which is observed as ovoidal structure in the cytoplasm ofthe capsulogenic cells. Then, it becomes bulbous in shape

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and appears trilaminar with a granulated dense coresurrounded by a medium dense electron opaque layer andan external lighter layer (Figs. 12, 13, 14, and 15).

In young polar capsules, the filamentous material stillexists in the core of the capsule (Fig. 13). In more advancedstages, the polar filament appears with two differentelectron dense zones, an inner dense and an outer lighter

one. In mature stages, the polar capsule elongates and thepolar filament becomes coiled several times and terminatesin a stopper and a capsular cap (Fig. 14). The capsularmaturation of all stages usually occurs synchronously(Fig. 13).

The external tubule is seen inside the cytoplasm of thecapsulogenic cells and is characterized by its electron dense

Fig. 1 Light micrographs.Squeezed cyst showingfresh spores of HenneguyasuprabranchiaeFig. 2 Light micrographs.Semithin section of theperiphery of a plasmodiumshowing the cyst wall and thedifferent stages (asterisk)Fig. 3 Light micrographs.Semithin transverse sectionof a spore showing the two polarcapsules (PC) and the sporo-plasm cell (Sc)Fig. 4 Light micrographs.Mature spore showing the sporebody (SB) with two polarcapsules (PC) and the tailregion (T)Fig. 5 Light micrographs.Semithin longitudinal section ofmature spores showing the body(SB) and the spore tail (T)

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core of granulated materials. This tubule is surrounded by agirdle of microtubules and disappears as soon as the polarfilament is formed within the capsules (Figs. 11, 12, 13, 14,and 15).

Valvogenesis

Valvogenesis is initiated when valvogenic cells start tosurround the capsulogenic and sporoplasmic cells and

Fig. 10 Transmission electronmicrographs. Three-celledpansporoblast apparentlyresulting from the division ofthe sporont within the pericyteFig. 11 Transmission electronmicrographs. Five-celledpansporoblast consisting ofalready differentiated cells, twovalvogenic (VC), two capsulo-genic (CC), envelope cell (EC),external tube (ET), andsporoplasm (S)Fig. 12 Transmission electronmicrographs. Nearly maturespore with polar capsules (PC)containing the coiled polarfilament (PF)Fig. 13 Transmission electronmicrographs. Section throughtwo developing pansporoblasts,each in a separate envelopingcell (EC) showing capsulogeniccell (CC) with a polar capsule(PC) containing filament (PF).Valvogenic cells (VC) andbinucleated sporoplasm (S)with mitochondria (M) andglycogen (Gly)

Fig. 6 Transmission electronmicrographs. Unicellular stages[=generative cells (GC)] with alarge nucleus (N) and a promi-nent nucleolus (Nu). Note thepresence of mitochondrialstructures (M)Fig. 7 Transmission electronmicrographs. Unicellular stages[=generative cells (GC)] with alarge nucleus (N) and a promi-nent nucleolus (Nu). Note thepresence of mitochondrialstructures (M)Fig. 8 Transmission electronmicrographs. Two opposedgenerative cells beforeenvelopmentFig. 9 Transmission electronmicrographs. Two celledpansporoblast as results of theinclusion of the sporont (GC) bythe enveloping cell (EC)

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rapidly adhere from their ridges through the desmosomal-like junctions. The posterior end of the valves protrudesforming the primordia of the posterior process. Numerousmicrotubules are observed concentrated at the ridges andare distributed within the valvogenic cytoplasm (Figs. 14and 15). During sporoplasm maturation, several boundedelectron dense bodies (the sporoplasmosomes) appear.These are spherical in shape and scattered throughout thecytoplasm (Figs. 11, 12, 13, 14, and 15). In immature andmature spores, the valves appear uniformly smooth withslightly thickened ridges joined in a suture line andprotrude posteriorly into two posterior processes. Eachmature spore has a spore body and two posterior processes

and surrounded by a homogeneous sheath which covers thespore (Figs. 16 and 17).

Discussion

Light microscopy

With respect to morphology and size of the spores, severalspecies of the genus Henneguya are found to resemble thepresent form: H. clariae Abolarin, 1971 infecting the gillsof Clarias lazera in Nigeria; H. shaharini Shariff, 1982infecting the gills of the marble goby Oxyeleotris marmoratiis

Fig. 14 Transmission electron micrographs. Longitudinal sectionthrough a nearly mature capsulogenic cell (CC), containing a nucleus(N) and a polar capsule (PC). Within each polar capsule occurs acoiled polar filament (PF). Only one nucleus (N) is seen. Theaccumulation of valve forming materials (VM) is observed in thesporoplasm of the valvogenic cells. The apex of the pyriform capsuleis plugged by the polar stopper (St)Fig. 15 Transmission electron micrographs. Longitudinal sectionthrough a nearly mature capsulogenic cell (CC), containing a nucleus(N) and a polar capsule (PC). Within each polar capsule occurs acoiled polar filament (PF). Only one nucleus (N) is seen. The

accumulation of valve forming materials (VM) is observed in thesporoplasm of the valvogenic cells. The apex of the pyriform capsuleis plugged by the polar stopper (St)Fig. 16 Transmission electron micrographs. Section through the basalportion of the spore body showing its continuity with the tail (T). Thespore is externally surrounded by a homogeneous sheath (arrows)Fig. 17 Transmission electron micrographs. Immature spore contain-ing two polar capsules (PC) and a single sporoplasm cell (SC)completely surrounded by a shell of two valves (VC). Note the denseappearance of the spore contents

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in Malaysia; H. bopeleti Fomena and Bouix, 1987 infectingthe gills of the catfish Chrysichthys nigrodigitatus inCameroon; H. suprabranchiae Landsberg, 1987 infectingthe suprabranchial organ of C. lazera in Israel; H. branchialisAshmawy et al., 1989 infecting the suprabranchial organand gills of C. lazera in Egypt; and H. nyongensis Fomenaand Bouix, 1996 infecting the gills and muscles ofMarcusenius moorii in Cameroon. Comparing with thepresent material, H. shaharini and H. nyongensis differ intheir number of polar filament windings (H. shaharini=3 to7, H. nyongensis=4 to 5 vs. 10 to 11). Spores of H. bopeletidiffer from those of the present study in their larger size andstraight caudal processes. In addition, all the previouslydiscussed species were described from other hosts which aretaxonomically far from the present host species. H. clariaecan be easily distinguished from the present parasite byhaving a larger spore size and longer caudal processes. Theonly two species which could be identical with the presentspecies are H. suprabranchiae and H. branchialis. Morpho-logical and dimensional comparisons between the three formsresult in considerable similarities. The variation seen in sizebetween such forms may be referred to the degree of maturityof the spores. Therefore, the present species could beidentified as H. suprabranchiae, while H. branchialis canbe considered as a junior synonym of H. suprabranchiae.The present study revealed that H. suprabranchiae reportedbefore in the catfish C. lazera (Landsberg 1987) has a newhost, Oreochromis niloticus.

Electron microscopy

Plasmodia

The ultrastructural studies of the plasmodial wall ofmyxosporea are of major importance to show the charac-teristic features of this important group of parasites (Currentand Janovy 1976). The ultrastructure of the plasmodiumdiffers not only among the different species of Henneguyabut it also differs between the “clinical types” of a singlespecies (Current and Janovy 1978). The plasmodial wall ofthe present Henneguya species is composed of a singlemembrane and is covered by a fine granular coat preventingthe direct contact between the trophozoite and the hosttissue. This observation corresponds to H. branchialis(Ashmawy et al. 1989) and coincides with that reported inthe case of H. piaractus (Adriano et al. 2005).

As in other myxosporeans, usually the peripheralportions of the plasmodia of the present parasite were filledwith generative cells and young pansporoblasts, while inlater stages of spore development they were located morecentrally. Generally, young plasmodia contained relativelyfew mature spores, whereas in older (mature) plasmodiaimmature and mature spores were observed.

Mature spores of Myxosporea exhibited a very densecytoplasm surrounded by an extremely electron dense shelland provided with elongated polar capsules containingcompletely formed and identifiable polar filaments with nocapsulogenous or valvular nuclei. All other stages wereconsidered immature (Lom and de Puytorac 1965; Schubert1968; Lom 1969). Each spore of the present parasite hadtwo equal polar capsules with polar filaments and twovalves which agree in the general architecture with otherresults (Schmahl et al. 1989; Azevedo and Matos 1989;El-Matbouli et al. 1990; El-Mansy and Bashtar 2002;Matos et al. 2005).

Sporogenesis

Sporogenesis is an exceedingly complex process involvingseveral cells which undergo distinct differentiations withinanother cell (envelope cell) (Wolf et al. 1986; El-Matbouliand Hoffmann 1989). In this study, early sporogenesisbegan when a sporont was engulfed by an enveloping celland lasted until the cell divisions had terminated. Lom andde Puytorac (1965) found that the pansporoblast inmyxosporeans forming large polysporic plasmodia devel-ops from a union of two generative cells; one envelopes theother (being the enveloping cell of the pansporoblast),while the enveloped one is in a process of proliferation anddifferentiation. These findings agree with many ultrastruc-tural studies of sporogenesis in several myxosporean generasuch as Henneguya, Myxidium, and Myxobolus (Listebargerand Mitchell 1981; Pulsford and Matthews 1982; Lom et al.1986). Moreover, the enveloping cell of some myxosporeanspecies is morphologically distinct from the sporont(Current et al. 1979; Pulsford and Matthews 1982; El-Mansy and Bashtar 2002; Matos et al. 2005).

Consequently, since Lom et al. (1982) concluded that theorigin of the pansporoblast occurs by the union of twogenerative cells (the enclosed state), it can be supposedthat myxosporeans form large polysporic plasmodia. Theyadded that the non-dividing pericyte played an importantrole in compartmentalizing the differentiating sporontand in supplying structural support during sporogenesis.Nevertheless, the pericyte may be instrumental in severalother aspects of this complex process, involving severalcells which undergo differentiation, all within another cell.It is possible that the pericytes supply essential nutrientsto the sporont progeny and that they may also supplymolecular regulators which control sporogenesis (Currentand Janovy 1978; Current et al. 1979; Abdel-Ghaffar et al.2005) and all these characteristics and events are inagreement with the presented results herewith.

The late sporogenesis begins when the cells arrangethemselves into their final position prior to spore con-structions. Structural differentiation starts as soon as two

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valvogenic cells surround a diplokaryotic sporoplasm andtwo capsulogenic cells and finally give rise to a maturespore. Cell differentiation as well as the arrangement of thedifferentiated cells has been reported to occur very rapidlyin several myxosporean species (Lom and de Puytorac1965). In the present investigation, only one spore wasformed within a pansporoblast. It was revealed that eachspore-producing unit in H. suprabranchiae consisted oftwo capsulogenic cells and a sporoplasm cell surroundedby two valvogenic cells and gave rise to a mature sporewith two polar capsules and an infective sporoplasmsurrounded by a shell of two valves. These results agreewith many reports (Current et al. 1979; Schmahl et al.1989; Azevedo and Matos 1989; El-Matbouli et al. 1990).

Capsulogenesis

The polar capsules as described by Cheissin et al. (1961)are spherical or ovoid in shape, contain the coiled polarfilament and their walls, and were continuous with thewalls of the capsular primordium. Also, Lom and Vavra(1963) found that the suggested double filament in the polarcapsule of H. psorospermica was in fact a single spirallytwisted filament. Regarding the sequence of the capsulo-genic events in H. suprabranchiae investigated herein,there are virtually similar to those reported for othermyxosporean species (Schubert 1968; Lom 1969; Currentand Janovy 1978; El-Matbouli et al. 1990). Microtubularparticipation in capsulogenesis of several myxosporidianswas suggested (Lom 1969; Current and Janovy 1978;Desser and Paterson 1978). They added that intracellularmovements and contractions are involved in the polarcapsule formation due to the fact that microtubules havebeen implicated in the movement of a wider variety ofmolecular and macromolecular components in the cells(Williams and Wolff 1970; Wessels et al. 1971).

In the present study, the filament appeared early in theexternal tubule while the primordium was still filled withgranules without any traces of filament. Similar results weregiven by other investigators (Lom and de Puytorac 1965).The rudimentary polar filament is formed within thecapsular primordium and the external tubule which possiblyhas its origin in the dense granular material present in bothstructures (Desser et al. 1983). On the other hand, it isgenerally thought that the external tubule invaginates intothe polar capsule and undergoes reorganization to becomethe polar filament (Lom and de Puytorac 1965; Schubert1968; Desser and Paterson 1978; El-Mansy and Bashtar2002; Matos et al. 2005).

The development of polar capsules of H. suprabranchiaein the present investigation was usually asynchronous.Similar findings were obtained by other investigators(Desser and Paterson 1978; El-Matbouli et al. 1990). In

addition, mature polar capsules of the present materialwere banana-like or pyriform in shape which is similar toseveral myxosporeans (Schubert 1968; Lom 1969; Currentand Janovy 1976, 1978; El-Mansy and Bashtar 2002;Matos et al. 2005).

Valvogenesis

In most bivalvulid species including the present myxospor-ean species, the orientation of the junctions of valvogeniccells (manifested as the sutural line) is set in a planeperpendicular to the spore circumference. In Myxosporea ingeneral, microtubules are not only involved in the polarcapsule formation, but they are also observed in thevalvogenic cells during sporogenesis. As valvogenesisproceeds, several microtubules appear within the cytoplasmof the valvogenic cells, especially in the valve suture-forming regions (Current and Janovy 1976, 1978; Hulbertet al. 1977; Current et al. 1979; Desser and Paterson 1978;El-Mansy and Bashtar 2002).

Sporoplasm

As a prominent and characteristic feature of myxosporeansporoplasm, many spherical electron dense bodies called“sporoplasmosomes” are scattered throughout the cyto-plasm (Schmahl et al. 1989; Azevedo and Matos 1989; El-Matbouli et al. 1990). These sporoplasmosomes exhibit agreat diversity in size but nothing is known on theirfunction. In the present investigation, many sporoplasmo-somes were observed inside immature and mature spores.The sporoplasmosomes of this parasite have a unique shapeand contain electron dense inclusions. As sporoplasmmaturation proceeds, there is an increase in the number ofmitochondria and an accumulation of glycogen particlessimilar to those reported for several other myxosporidianspecies (Current and Janovy 1976, 1978; Current et al.1979; El-Mansy and Bashtar 2002; Matos et al. 2005).These authors suggested that maturation of sporoplasm“germ cell” involved storage of metabolic reserves andacquisition of aerobic metabolism which, under the properstimulus, could provide energy necessary for the establish-ment of the sporont within a new host.

Acknowledgement The authors are thankful for the funding supportof Center of Excellence for Biodiversity Research, College of Science,King Saud University, Riyadh, Kingdom of Saudi Arabia.

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