Apolocystis perienteron sp. nov. (Apicomplexa: Monocystinae) a new species of aseptate gregarine from Pheretima californica (Annelida: Oligochaeta) from Egypt.
An acephaline gregarine, Apolocystis perienteron, was described as a new species from the coelomic cavity and intestinal coelomic epithelium of earthworm, Pheretima californica. The young trophozoites are intracellular, found inside the coelomic epithelium of the intestine; while the adult trophozoites (48 -72.5 µm in diameter) is found free in the coelomic cavity or mostly attached to either somatic or splanchnic epithelium. Gametocysts are 76-150 µm in diameter. Navicular sporocysts measured 17 x 10 µm, with slightly projected flat plugs. Histological and ultrastructural details of the trophozoite and gametocyst were described.
Keywords: Gregarine, Pheretima, coelom, Apolocystis.
Over 400 species of aseptate gregarines were described in order Eugregarinida (Levine 1977). Trophozoite of aseptate gregarine is formed from one cytoplasmic part. The significance of the gregarines from evolutionary view is due to their supposed early diverging position into the group of apicomplexa and having many characteristics presumed to be plesiomorphic (Théodorides 1984; Vivier & Desportes 1990 and Cox 1994). The genus Monocystis stein, 1848 (Order: Eugregarinida) was described by Hesse (1909) and contains four genera, namely, Monocystis, Nematocystis, Rhynchocystis and Pleurocystis. Genus Apolocystis was recognized by Cognetti de Martti (1923) to hold all species of Monocystis which had spherical trophozoites with no polarity. Many authors had described several species of Apolocystis from wide regions of the world (Bahatia and Setna 1926; Phillips and Mackinnon 1946; Ramadan, 1969; Levine 1977; Segun 1978; Pradhan and Dasgupta 1983; Armendáriz and Gullo 2002; Bandyopadhyay et al. 2004 and Bhowmik et al. 2012). In this work, a new species of Apolocystis is described with a comparative discussion with closely related species.
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MATERIALS AND METHODS
The host worms
Worms were collected from zoological gardens, Giza governorate. Worms were then put into soil-filled plastic containers and transferred alive to the laboratory of invertebrates, zoology department, faculty of science, Ain shams university. Some worms were dissected and the seminal vesicles were carefully removed and placed on a clean grease-free slide with a drop of 0.8 % NaCl solution. A thin film of the seminal fluid was drawn out on the slides which then covered with glass cover slips. Slides were examined for living protozoans under a light microscope. The coelomic fluid of the examined worms was withdrawn by a micropipette and then placed on clean slides with a drop of 0.8% NaCl solution; slides were then covered with glass cover slips and examined. After the initial examination of living protozoans, slides were semidried and fixed for 20 min. Schaudin’s fluid (saturated aqueous solution of mercuric chloride and glacial acetic acid). Slides were then stored in 70% ethyl alcohol for removal of excess of mercuric chloride.
Sectioned materials were prepared by taking body segments of infected worms and then placed in Bouin’s fluid fixtive for 24 h. Specimens were then transferred into a mixture of 70% ethyl alcohol and lithium carbonate (for removal of yellowish colour of Bouin's fluid). Specimens were then passed through an ascending series of alcohols (1h. for each change), and then tissues were cleared in terpineol for 3 days. Specimens were then embedded in wax, sectioned (5 µm thickness) on clean slides; the slides were then deparafinized using xylene. The slides were then passed through ascending series of alcohols (2 min. for each change) and finally they were placed in distilled water. Some slides were transferred to 3% iron alum solution (3 h) and stained with Heidenhain’s haematoxylin solution (20 min.). Differentiation was done with1% iron alum solution under the low power objective lens of a compound microscope. The slides were then washed thoroughly with distilled water, dehydrated in an ascending series of alcohols, cleared in xylene and mounted in Canada balsam. Other groups of prepared slides were placed in haematoxylin stain for 30 min; then slides were transferred to a tap water (1 min.), and then were dipped in eosin stain, finally slides were dehydrated in an ascending series of alcohols, cleared in xylene and mounted in canada balsam. Photomicrographs were taken by a Kodak digital camera (model 1450Z) attached to a compound microscope.
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Infected worms' parts were washed in 0.8 % NaCl then fixed in a 2.5 % glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.4) for 24 h. The specimens were then washed in a phosphate buffer solution (2 h.); a postfixation treatment was done by 1% osmium tetraoxide (Os4) followed by washing twice in a distilled water. Samples were dehydrated, embedded in resin, ultrasectioned by ultramicrotome, placed on copper grids and stained with uranyl acetate (15 min) and then lead citrate (5 min), then grids were examined by a JEOL’s electron microscope (JSM-6300) at the regional center of fungi, El Azhar University, Cairo.
Apolocystis perienteron sp. nov. (Figs. 1-15; Tables 1 - 3)
Phylum: Apicomplexa, Levine 1977
Order: Eugregarinida, Leger 1900
Family: Monocystidae, Butschli 1882
Subfamily: Monocystinae, Bhatia 1930
Genus: Apolocystis, Cognetti de martiis 1923
Description: Sub-spherical, oval, intracellular young trophozoites (inside the coelomic epithelium of the intestine). The adult trophozoites (48 -72.5 µm in diameter) have neither polar differentiation nor ectoplasmic processes. Trophozoites were mostly attached around the intestine of the host. Gametocysts were 76-150 µm in diameter. Navicular sporocysts measured 17 ± 2 x 10 ± 0.6 µm, with slightly projected flat plugs.
Remarks: Young trophozoites were intracellular, found in coelomic epithelium of the intestine (Fig. 1), while developing and adult trophozoites (Fig. 2) and gametocysts were found in coelomic cavities of middle portion of the infected worm. Histological sections obviously shows that most stages of the parasite were surrounded by amoebocytes of the host (Fig. 3), these amoebocytes assisting in attachment of most life cycle stages to coelomic epithelium of the intestine and to that of the body wall of the infected worm (Fig. 4). Electron microscopy indicated that the parasite-attached amoebocytes were linked with different sites of the host tissues by highly branched processes protruded from amoebocytes in direction of the nearest host tissue (Fig. 5). On the other hand, the trophozoite, which was surrounded by amoebocytes, has epicytic extensions; these extensions were observed in two forms: the first one was a single extension (15 µm in length) developed in a direction closely parallel to the circumference of the trophozoite (Fig. 6), the second one is represented by pairs of straight extensions (6 µm in length for each). Both forms are not only running closely parallel to the circumference of the trophozoite but they also are opposite to each other, acting like pliers (Fig. 7). The tops of the epicytic extensions were pointed, few in their number and had no characteristic pattern in their distribution around the circumference of the trophozoite. These epicytic extensions contribute in tight attachment of trophozoite with surrounding amoebocytes. In some semithin sectioned materials, there are continuous chromatophilic irregular patches accumulated below the cell membrane of the trophozoite which then partially separated as scattered patches into the cytoplasm and finally accumulated around the outer surface of the nuclear membrane (Fig. 8). By electron microscopy, these patches transformed into moderately electrodense fuzzy material which located below the cell membrane of the parasite (Fig. 9), while those in the cytoplasm were interspersed by highly osmiophilic spherical granules of various sizes (Fig. 10). The adult trophozoites, which had no polar differentiation, measured about 48 - 72.5 µm in diameter, (Table 1).
The nucleus was spherical, showed no fixed position (9-18.5 µm in diameter), with a spherical, eccentric, vesicular endosome (4-7 µm). Electron microscopical studies showed that the nucleus undergoes a periodical activity in its chromatic materials, in some trophozoites'
the chromatin material appeared in the form of an osmeophilic continuous thin layer below the nuclear membrane and extended toward the karyosome in the form of separate scattered patches into the nucleoplasm (Fig. 10). In other cases, these patches were not observed and the nucleoplasm was coarsely granulated (Fig. 11) and the nuclear membrane was clearly observed, wavy and discontinuous at intervals where the nucleopores were present (Fig. 12). In other areas of the nuclear membrane, the wavy nature of nuclear membrane disappeared (Fig. 13). Besides, electron-dense round bodies may present inside the nucleoplasm (Fig. 11).
The paraglycogen granules were round to ovoid in shape, having a strong reaction to PAS; the diameter of these granules ranged from 0.6 to 1.7 μm in length, with an average of 0.9 ±0.7 μm. Gametocysts were observed in the coelomic cavity, with sizes ranged from 76 to 150 μm in minor diameter and from 114 to165 μm in major diameter. Ultrastructurally, the cyst wall of the gametocyst was composed of two layers: one outer thick moderately electrodense fuzzy layer, about 445 nm in thickness, the other is an inner highly electrodense, measured 35 nm in thickness (Fig.14). Sporocysts were navicular, with projecting flat plugs in both ends, and were 10 ± 0.6 µm in width and 17 ± 2 µm in length (Fig. 15).
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In Egypt, Ramadan (1969) described Apolocystis aggregata from the seminal vesicles of Pheretima caliphornica and P. hawayana. In table 2, although the measurements of the nuclei and the karyosomes related to Apolocystis aggregata fall in the range of those of the species under consideration, the two species showed some different features, the trophozoite of A. aggregata is larger, the young trophozoites of A. aggregata aggregate in small groups consisting of 5 to 20 individuals, the size of the gametocysts of A. aggregata is larger than that of the present parasite, spores of A. aggregata are larger in size with pointed ends, while those of our species have small blunt ends. Ramadan (1969) stated that; in A. aggregata an amoebocyte layer is formed only around each young stage, while in the present study, this phenomenon is common in all stages until before secreting the gametocyst wall around the two syzygites (gamonts).
Some gregarines described in other countries, exhibited some features which may be comparable with those of our present species. Some species of Apolocystis perform completely or partially their life cycle in the host coelomic cavity such as: A. catenata Muslow, 1911; A. michaelseni Hesse, 1909, and A. janovyi Armendariz and Gullo, 2002. Trophozoites and gametocysts of A. catenata were characterized by the hugeness of their sizes (425 and 500 μm in diameter, respectively) and their sporocysts were smaller than those of our present parasite (Table 3).
Despite the similarities in many aspects between A. michaelseni and A. perienteron n.
sp. (Table 3), there are some sharp differences like: the trophozoites and gametocysts described in the present work are markedly smaller in sizes than those of A. perienteron n. sp. and the number of the sporocysts in the gametocyst of A. michaelseni was 16 at maximum, while in A. perienteron they were numerous, finally, a swollen part in the equatorial plane of the sporocyst of A. michaelseni was described which not reported in A. perienteron.
Armendariz and Gullo (2002) reported the presence of basophilic granules in the cytoplasm of the trophozoite of A. janovyi, measuring about 0.62 μm to 1.8 μm, and increasing in size towards the nucleus where they appeared to be more dispersed. Comparable intra endoplasmic irregular chromatophilic patches were observed in the trophozoite of the species under study. These masses were, in most cases, under the pellicle of the trophozoite and randomly scattered in the endoplasm, besides, Armendariz and Gullo reported that young trophozoites were intercellular (in which species), located near the basal membrane of intestine, while in the present study, A. perienteron were intracellular in visceral peritoneal cells included only in post-clitellar region of the worm. Martinucci and Crespi (1979) described filiform extensions from surface of trophozoites of a species of Apolocystis which were comparable with similar epicytic extensions in the trophozoite of A. perienteron. Martinucci and Crespi also stated that the maximum length of these cylindrical extensions was 6 μm and their diameter varied between 0.2 to 0.3 μm and supported by microtubules, in A. perienteron two forms of epicytic extensions were observed: a long single extension, 15 μm long and 0.4 μm to1 μm wide and paired extensions, each 6 μm long and no internal microtubules were observed into these extensions.
Martinucci et al. (1981) investigated the ultrastructure of the gamont in syzygy, of gametes and zygotes in Apolocystis sp. He stated that wall of the gametocyst was formed by one layer (50 nm thick) made up of two parts: outer strongly osmiophilic and inner moderately electrodense part, In contrast, in A. perienteron, the gametocyst wall was composed of two layers: an outer thick moderately electro densified fuzzy layer (445 nm thick) and an inner thin highly electro densified one (35 nm thick). Thus it is clear from the above detailed comparison that our species in question differs from the previously known species of the genus Apolocystis justifies declaring it as a new species of Apolocystis, namely; A. perienteron n. sp. as it is always found surrounded by amoebocytes and mostly attached to epithelial layer of the intestinal region (enteron) of the host worm.