Apolocystis nephredii (Apicomplexa: Monocystinae) A New Aseptate Gregarine Species from Nephredia of Limnodrilus sp. (Annelida: Oligochaeta) from Egypt

The Journal of Medical Entomology and Parasitology is one of the series issued quarterly by the Egyptian Academic Journal of Biological Sciences. It is an important specialist journal covering the latest advances in that subject. It publishes original research and review papers on all aspects of basic and applied medical entomology, parasitology and host-parasite relationships, including the latest discoveries in parasite biochemistry, molecular biology, genetics, ecology and epidemiology in the content of the biological, medical entomology and veterinary sciences. In addition to that, the journal promotes research on the impact of living organisms on their environment with emphasis on subjects such a resource, depletion, pollution, biodiversity, ecosystem.....etc.


INTRODUCTION
Aseptate gregarine fauna has been noted in many areas of the world including Egypt.Gregarine fauna of Egypt needs more investigations to explore the new species.About 400 species of acephaline gregarines were described in order Eugregarinida (Levine 1977).Trophozoite of aseptate gregarine characterized by a single cytoplasmic part.
Genus Apolocystis was documented by Cognetti de Martti (1923) to gather all species of Monocystis contain spherical trophozoites with no polarity.Many studies identified several species of Apolocystis from various regions of the world (Bhatia 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 and2012;Ramadan et al. 2014 and2015).In this paper, taxonomic description of a new species of Apolocystis is given and its phases of life cycle described.

MATERIALS AND METHODS Host Worms:
Three hundred and twenty-four worms collected from both Maghagha, and Abo-Rawash regions, Menia and Giza Governorates respectively, Egypt.Worms were put into freshwater-filled plastic containers and then transferred alive to the laboratory of invertebrates, Department of Zoology, Faculty of Science, Ain Shams University.Worms were kept alive for days in small pools of dechlorinated water, Water was changed daily.Some worms squeezed between two clean slides and slightly compressed to observe the parasite inside nephredia.Other worms were cut into small pieces in Petri dishes filled with saline solution (0.8 % NaCl), to free the parasite stages.Carefully, living parasites removed and placed on a clean grease-free slide with a drop of saline solution to be exanimated under a compound microscope then photographed.After the initial examination of living protozoans, slides were semi-dried and fixed for 20 minutes in Schaudin's fluid (Mercuric Chloride and Methanol).Slides were then put in ethyl alcohol for removal of excess of mercuric chloride.One set of specimens were prepared for staining by Heidenhain's haematoxylin and Haematoxylin and eosin as described in Ramadan et al., (2014).

Histological Preparations:
Sections (5µm) were prepared by taking small pieces of infected worms that fixed in Bouin's fluid for 24 hours, finally, section were stained by Haematoxylin and eosin and Giemsa's stain, dehydrated, cleared and mounted.Photomicrographs were taken by a Kodak digital camera (model 1450Z) attached to the compound microscope.This parasite was detected in the nephredia of Limnodrilus sp. (Fig. 1A).From 324 collected worms, 17 (5.2%) were found infected with Apolocystis nephredii.All 17 worms only infected with this parasite.The intensity of infection was higher in the nephredia of the middle segments and getting lighter toward the posterior segments.Syzygy and gametocyst stages were the most observed stages in the nephredia.

RESULTS AND DISCUSSION
The young trophozoite was typically round in shape (Fig. 1B).The nucleus of the early stages was relatively large, rounded, centrally located (Fig. 1B) and usually occupied a considerable space inside the young trophozoite.In sectioned material stained with haematoxylin and eosin, darker red chromatin granules appear in nuclear space (Fig. 1B).A single rounded dense centric karyosome was also observed (Figs.1B, 1C & 1D).Many chromatin vacuoles were observed in the periphery of karyosome, these vacuoles were more or less equal in size but their distribution inside the karyosome took either a regular or irregular modes (Figs.1C, 1D & 3A).
The adult trophozoite was pear-(Fig.1E), kidney-or oval-shaped (Figs.1F & 3B), surrounded by the remnant parts of the nephredial wall.The dimensions of adult trophozoites ranged between 75 -85 x 81-124 µm, with an average of 79 (±3.7) x 92.7 (±24) µm, Moreover, the endosarc was filled with round to oval paraglycogen granules, ranging between 1 and 1.3 µm, with an average of 1.1 (±0.1) µm.The nucleus in most adult trophozoites, tended to be eccentrically situated within the parasite's body, the nucleus ranged between 19 and 24 µm, with an average of 22.4 (±2.1) µm.In fresh preparations, most of the nuclear space between the nuclear membrane and the karyosome was clear and homogenous.The internal surface of the nuclear membrane had a discontinuous layer of large dark dot-like chromatin material, which was more or less regularly distributed under the circumference of the nuclear membrane (Figs.1E & 3C).The average dimensions of the recently formed syszygy stages were 190 (±13.7)x 117 (±14.4)µm.The mature gametocysts were mainly oval filled with numerous mature sporocysts (Figs.2B & 3D).Generally, the gametocysts measured 200 -219 x 112 -132 µm, with an average of 208.9 (±9.8) x 125.1 (±4) µm.
Sporocysts were navicular with a thickened flat region at the apical end measuring about 2 µm (Figs.2C & 3E).Generally, the sporocysts measured 17 -19.2 x 8.1 -9 µm, with an average of 18.5 (±0.6) x 8.5 (±0.2) µm.Ramadan (1969) described some acephaline gregarines isolated from different species of Pheretima, P. californica, P. hawayana and P. elongata, which are belonging to order Opisthopora, family Megascolecidae and two species of Alma, belonging to family Glossoscolecidae in Egypt.The host of this parasite under consideration, genus Limnodrilus, was belonging to family Tubificidae, so the gregarines parasitizing genus Alma could be comparable with our parasite recorded in genus Limnodrilus.Ramadan (1969) reported two species of Apolocystis, A. almanili and A. centrospora from the seminal vesicles of two Alma species.
According to Table 1, the measurements of the trophozoites and their nuclei belonging to A. almanili were slightly larger than those of corresponding stages in the parasite under investigation.Ramadan (1969) described the paraglycogen granules as following: "The preparations stained with iron-alum haematoxylin the endoplasmic granules take a faint green color, which is a characteristic feature of this parasite" this feature was not observed in the endoplasm of the parasite under question.In fresh state, the nucleus of A. nephredii, exhibited a discontinuous layer of dot-like chromatin more or less regularly distributed under the nuclear membrane, while in stained materials there is an irregular chromatin layer under the nuclear membrane with thin strands of chromatin spreading toward the karyosome until fused around the karyosome.In the description of A. almanili, Ramadan (1969) also stated that the nucleoplasm contains scattered fine chromatin granules except at the small area around the karyosome that is clear, unstained and devoid of any granulation.
The size of the gametocyst in A. almanili was markedly smaller than that of the present parasite.Besides, Ramadan reported a high degree of shrinkage of the gamonts after encystment leading to a wide space left between gamonts and the cyst wall, this space may reach about 50 µm.This aspect was not observed in early gametocyst of the current parasite.
In Apolocystis almanili, Ramadan described three types of navicular sporocysts.In the parasite under question, only one type of sporocysts was reported with thickened flat ends measured 17-19.2x 8.1-9 µm, with an average of 18.5 (±0.6) x 8.5 (±0.2) µm which is closely related to the first type of Ramadan's gregarine.
Apolocystis centrospora differs from the present Egyptian monocystid in some aspects; A. centrospora had markedly smaller sporocysts, Besides, they, as described by Ramadan (1969), were filling the centers of their gametocysts only.Ramadan (1969) reported two forms of gametocysts of A. centrospora; a round form and an ovoid form.The present parasite resembled the last form but was slightly smaller in size than that of corresponding ovoid form of Ramadan's work.Finally, the size of the trophozoites of A. centrospora was twice in size, that of corresponding stages in the present parasite.(Table 1).
Among the previously known species of Apolocystis, Table 2 shows species fall in the range of size of the species under consideration.In A. beaufortii, the gamonts produce two morphologically distinguishable types of gametes (anisogametes).The sporocysts are biconical with short flat plugs and are markedly smaller than those of the parasite of Limnodrilus sp.In A. chotonagpurensis, trophozoites characterized by very fine rows of cytoplasmic ridges, the gametocysts are ellipsoidal and mainly smaller, in addition, the sporocysts are shorter and narrower than those of the parasite of Limnodrilus sp. in the present work.
A. granulata, the trophozoites have larger nuclei that may reach 50 µm.Bereczky (1967) recorded the presence of alveoli into which paraglycogen granules are located that cannot be observed in present parasite.
In A. saigonensis, the gametocysts are smaller in size than those of our parasite.The sporocysts are smaller with pointed ends.
Trophozoites of A. minima, have small nucleus (8 µm), the paraglycogen granules reach 3.2 µm in diameter.Boisson (1957) revealed to the presence of anisogametes in the life cycle of A. minima.The gametocysts and sporocysts are smaller in size.
Pradhan and Dasgupta (1983) described A. monokaryoseminiferus that collected from the seminal vesicles of Amynthas robusta, its trophozoites measured from 61-115 µm, the other stages of the life cycle were not described.
Only one species, belonging to Apolocystis which was mainly recorded in the coelom and occasionally in nephredia, Apolocystis michaelseni Hesse, 1909(Cognetti de Martiis 1923).The trophozoites are spherical or ovoid (up to 225 or 295 x 230 µm), have dark opaque appearance resulting from their content of "voluminous" granules, and their cytoplasm shows certain staining irregularities.The gametocysts of two gamonts are ellipsoidal (235-300 x 170-220 µm) and abundant, cysts containing one or three individuals.The mature gametocyst contains up to 16 sporocysts.The sporocysts of A. michaelseni are strongly swollen in the middle and are slightly smaller [15 x 9 µm] than those of the parasite of Limnodrilus sp.
The present species of Apolocystis is considered, universally, the second record of a gregarine, belonging to family Monocystidae (after) which is parasitizing, Limnodrilus sp.(Family: Tubificidae) and locally, the first record in Egypt.It should be stated that, according to Frolov (1991), Janiszewska (1968) was the first one who described first monocystid gregarine, Zygocystis limnodrili, from the vesicula seminalis of Limnodrilus hoffmeisteri.However, the present species is the unique species of Apolocystis which complete all life stages within a specific organ other than seminal vesicles, namely; nephredia.
Thus, it is clear from the above discussion that the gregarine recorded from Limnodrilus sp. is the first species of Apolocystis which infects this genus of the host worm.However, the present authors recommend giving a name to this new species of Apolocystis as A. nephredii n. sp.TAXONOMIC SUMMARY Description: Trophozoites are pear, kidney or oval in shape, with an average of 79 (±3.7) x 92.7 (±24) µm.The rounded nucleus has a single karyosome and measures 19 to 24 µm in diameter, with an average of 22.4 (±2.1) µm.Gametocysts are ellipsoidal, with an average of 205.9 (±3.8) x 125 (±4) µm.Sporocysts are navicular with flat thickened ends with an average of 18.5 (±0.6) x 8.5 (±0.2) µm.Etymology: the new species has been named after the site of localization within the host.

Legend of Figures
Figure 1:  A : A photomicrograph of the middle region of an infected worm showing the host's nephredia (arrowhead) highly infected with different stages of the parasite.Fresh preparation.B: A photomicrograph of a section of young trophozoite showing chromatin accumulation (arrowhead) under the nuclear membrane (Nm.)and central karyosome (K.).Haematoxylin and eosin.C: A photomicrograph of a section of a young trophozoite showing the chromatin vacuoles (arrowhead) inside the karyosome in one complete peripheral ring.Giemsa's stain.D: A photomicrograph of a section of trophozoite showing the irregularly accumulated chromatin vacuoles (arrowhead) in the peripheral region of the karyosome.Giemsa's stain.E: A photomicrograph of a trophozoite (T) inside intentionally ruptured nephredium (Nf.), Note: chromatin granules (arrowhead) under nuclear membrabe.Fresh preparation.F: A photomicrograph of a fully developed trophozoite, Haematoxylin and eosin.
Figure 2: A: A photomicrograph of a complete syzygy of trophozoites showing the hemispherical shape of gamonts.Heidenhain's haematoxylin stain.B: A photomicrograph of a gametocyst.Fresh preparation.C: A photomicrograph of mature sporocysts showing thickened flat regions in the apical ends (arrowhead) of the sporocyst.Fresh preparation.

Figure 3 :
Figure 3: Drawing of different stages of Apolocystis nephredii sp.nov.A: Enlarged nucleus of young trophozoite showing the vacuoles of different sizes inside karyosome B: Adult trophozoite showing its general shape.C: Nuclear details in an adult trophozoite showing a continuous layer of chromatin (arrowhead) under nuclear membrane (Nm).D: A gametocyst crowded by navicular sporocysts.E: A single sporocyst showing thickened flat region at the apical end (arrowhead).

Table 1 :
Comparison between the species of Apolocystis infecting Alma sp. and Limnodrilus sp. in Egypt.

Table 2 :
Comparison between the species of Apolocystis fall in the range of size of the species under consideration.