Light microscopy and surface topography of Urotrema scabridum and Renschetrema indicum ( Digenea ) from Rhinopoma hardwickii ( Chiroptera ) : first report in Egypt

This report introduced the description of two different species of digenean parasites isolated from the intestine of Rhinopoma hardwickii with new host and locality records in Egypt. The recovered helminthes were studied morphologically and morphometrically by light microscopy and the surface topography of the two species was elucidated by scanning electron microscopy (SEM). Urotrema scabridum had an elongated body, testes were tandem, ovaries were pretesticular, and vitelline follicles were observed in 2 lateral fields. SEM showed that the anterior half was covered with random and backwardly directed tegument spines. The lumen of the oral sucker was as a longitudinal slit encircled with type I dome-shaped papillae. The ventral sucker was wrinkled and covered by tongue-shaped tegument spines and several scattered papillae. Renschetrema indicum had a fusiform body with minute spines densely distributed in the anterior part of the body; testes sub-triangular, ovary fusiform; vitellaria were randomly distributed around the ceca and genital organs. SEM showed that the fore-body was ventrally concave and surrounded by cytoplasmic ridges equipped with numerous closely packed claw-shaped spines. The oral sucker was externally surrounded by two circles of papillae while the lip of the ventral sucker was rounded and surrounded by three papillae located in its upper end and anterolaterally.


Introduction
The tegument and its microtopographical features, such as papillae, spines and tubercles, are considered to be the major host-parasite interface and the most important taxonomical aspects of adult trematodes (MATA-LÓPEZ & LEON-REGAGNON, 2006).The tegument is involved in the protection of the parasite against host enzymes and immune systems, the excretion wastes, and the absorption of nutrients (BURTON, 1966;LUMSDEN, 1975;THREADGOLD, 1984;PAPERNA & DZIKOWSKI, 2006).Electron microscopic techniques, such as scanning (SEM) and transmission electron microscopy (TEM), are of a particular interest in the description of tegumental structures.Indeed, SEM can provide data on the external morphology and localization of these structures, while TEM allows for ultrastructural investigations at a cellular level.
From the parasitological point of view, bats are of a special interest for number of reasons: bats are known to be susceptible to many infections of animals and man.Although each species of bat has its own ecology and behaviour, a remarkable degree of interspecies associations occurs.These associations provide a good opportunity for the transmission of certain parasites between individuals of different species of bats (WIMSATT, 1970).
Little is known regarding the helminth parasites infecting Egyptian bats, with no available data in the literature since the study of Saoud & Ramadan (1976); they reported a numerical data on four species of digenean parasites without morphological identification, these were Prosthodendrium sp., Anchitrema sp., Lecithodendrium sp. and Acanthatrium sp. which needs further revision.So, these animals should attract the attention of several parasitologists working in Egypt as recommended by several authors who stated that the helminth fauna of bats in Egypt is fairly rich and nothing was done on the various aspects of the host parasite-relationships, particularly host specificity, the prevalence of parasites and the inter-relationships between members of the parasitic fauna.
Renschetrema species are digeneans of the order Plagiorchiida, family Renschetrematinae Yamaguti, 1971.These trematodes are parasites of vertebrates and are characterized by their pyriform body and the presence of spinose tegument, testis opposite in the hind body, vitellaria posterior to the testis and the uterus confined to the hind body.In the present study, the morphological and morphometric characteristics of two digenetic trematodes isolated from the intestine of the lesser mouse-tailed bat Rhinopoma hardwickii (family Rhinopomatidae) were investigated by light microscopy.Additionally, the surface ultrastructure of the two parasites was observed and compared by scanning electron microscopy.

Materials and Methods
Ten specimens of the lesser mouse-tailed bat Rhinopoma hardwickii (Chiroptera: Rhinopomatidae) were captured using a mist net from buildings where bats may found in Cairo, Egypt.A cotton ball soaked with isofurane was placed inside a plastic bag containing the bat and left inside to allow sufficient time for the anaesthetic gas to cause euthanasia.Alternatively, the bat was left in the sealed bag for sufficient time to achieve anaesthesia, at which time it was removed from the bag and injected intraperitoneally with an overdose of sodium pentobarbital.This finding is in agreement with the regulatory laws regarding experimental ethics of animal use and collecting permits, Institute of Animal Care and Use Committee (IACUC), protocol number CUFS/S/Para/36/14.Following dissection, trematodes were recovered from the intestine, immediately fixed and held in 10% neutral buffered formalin for 48 h, placed overnight in distilled water, stained overnight in acetocarmine, dehydrated in ethanol series, cleared in clove oil, and permanently mounted on glass slides using Canada balsam.Photomicrographs of stained whole mounted specimens were made with a Zeiss Axiovert 135 photomicroscope equipped with a PowerShot Canon digital camera.Drawings were made with the aid of a drawing tube.All measurements are presented in millimetres unless otherwise stated and are given as the range followed by the mean in parentheses.For scanning electron microscopy, specimens were fixed in 4% buffered glutaraldehyde, washed in cacodylate buffer, and dehydrated in alcohol.After passing through an ascending series of Genosolv-D, they were processed in a critical point drier "Bomer-900" with freon 13, sputter-coated with gold-palladium in a Technics Hummer V, and examined with an Etec Autoscan scanning electron microscope at 20 kV Jeol.

MORPHOLOGICAL DESCRIPTION
Urotrema scabridum Braun, 1900 Description (n=10): Light microscopic examination (Figure 1a-c) showed that the parasite had an elongated body 1.50-2.22(2.06±0.02)x 0.461-0.682(0.521±0.002) mm (maximum width at its middle).The parasite's suckers were powerful and spaced at a distance of 1.2 mm apart, the oral sucker was 0.162-0.284(0.19±0.002) x 0.230-0.502(0.341±0.002) mm.An intestinal bifurcation occurred just underneath the oral suckers.Pharynx 0.021-0.33(0.026±0.002) mm followed by oesophagus which  2 a-f ) of the described parasite (n=5) showed that the anterior half of the body was covered by backwardly directed tegumental spines regularly arranged in rows and decreasing in number going posteriorly.The ventral pit was at the posterior level of the middle one-third of the body.The oral sucker contained a terminal mouth which was longer than its width.The lumen of the oral sucker was in the form of a longitudinal slit, and the lip was devoid of tegumental spines.The lumen of the ventral pit was covered with spines, which were smaller than those around the whole body.Two sizes of type I dome-shaped papillae encircled the lip of the oral sucker.The lip of the ventral sucker was wrinkled and covered by tegumental spines and few scattered papillae.A line diagram for the ventral view of the parasite holotype is illustrated in Figure 3.

Remarks
The recovered parasite possessed most of the morphological features characterizing the genus Urotrema: the terminal genitalia is at the posterior part of the body; tandem testes near the posterior end of the hind body; an ovary in the anterior hind body; unlobed gonads; and vitelline follicles extending anteriorly or anterior to the level of the ventral sucker.Compared with some of the previously reported species from different hosts, U. scabridum Braun, 1900 is the most closely similar species; both have ceca that extend posteriorly behind the posterior testis and the vitelline follicles do not extend to the level of the posterior margin of the pharynx.Minor differences that are observed in the morphometric data between the two species may be related to the effects of specimen preparation (Table 1).

Taxonomic summary
Family: Urotrematidae Host: Egyptian lesser mouse-tailed bat Rhinopoma hardwickii.Locality: Attics of Egyptian buildings.Infection site: Intestine.Prevalence: four out of ten (40%) were naturally infected with this parasite alone, while three specimens (30%) showed a mixed infection with R. indicum.

Renschetrema indicum
SEM (Figure 4a-e) of the described parasite (n=5) showed that the fore body was ventrally concave, surrounded by cytoplasmic ridges equipped with numerous closely packed claw-shaped spines whose density decreased at the posterior extremity.As observed in all of the described specimens, the oral sucker and mouth opening were directed anteroventrally and externally surrounded by 12 papillae arranged as two circles (4 pairs located in the outer circle and 2 pairs located in the inner circle at each side of mouth).The ventral sucker was rounded and pre-equatorial, and its lip was surrounded by 5 papillae (3 located in the upper part of sucker and 2 located anterolateral, one at each side).Particular arrangements and variations in the morphology of spines were observed over the whole surface of the body, where the area around the oral and ventral sucker and in between contained tongue shaped tegumental spines differing from those around
Materials deposited: Parasitology division, Zoology department museum, Faculty of science, Cairo University, Egypt.

Remarks
This trematode is assigned to the genus Renschetrema (Microphallidae) according to the key published by Kifune (1984), which records only four species of this genus to date (Table 2).These species are Rohdetrema sandoshami Rohde, 1964 from Tylonycteris sp. and Kerivoula sp., Malaysia (Malay); Renschetrema malayi Rohde, 1964 from Rhinolophus sp. and Miniopterus juligino Kuala Senyul, Kelantan, Malay; and R. indicum Kifune, 1984 from Myotis nipalensis and M. muricola, West Bengal, West Sikkim India and Renschetrema sp. from Glischropus tylopus, Malaysia (Malay) (ROHDE, 1964).The present species differs from R. sandoshami in having an almost circular body, which is slightly broader than long; the vitellaria are extremely developed and distributed peripherally in contrast to the long body in the previously recorded parasite; it also differs from R. malayi whose two testes are remote from each other by at least a distance equal to their own breadth, while the testes are closely situated to each other in the present species.Renschetrema sp.differs by the presence of small vitelline follicles sparsely distributed in the preacetabular region in contrast to the large, compactly distributed vitelline follicles in the preacetabular region.However, the present parasite closely resembles R. indicum in most of the morphological features reported in the description of Kifune (1984) with some morphometric features somewhat less different.

General organization of the tegument
According to Halton & Maule (2004), microscopy has providing fundamentally important information on the structural and functional correlates of a number of key organ systems of helminths, such as their body surface.Detailed studies on the ultrastructural characteristics of digeneans can provide useful information in the taxonomic study of the groups.There are no existing records describing the surface topography and the ultrastructural characteristics of the tegument for the two species described herein.
The tegument organization of the two species is similar to the general tegument structure described for digeneans studied previously (MORRIS & THREADGOLD, 1968; BENNETT &  THREADGOLD, 1975;THREADGOLD, 1984;ROBERTS & JANOVY, 2000).Folds in the tegument such as cytoplasmic ridges on the body surface and cobblestone-like zones could participate in nutrition by amplifying the free surface area (PAPERNA & DZIKOWSKI, 2006).Cytoplasmic ridges of the tegument have also been observed in SEM micrographs of other digeneans, such as Bucephalus anguillae (FILIPPI et al., 2010), Fasciola hepatica (BENNETT, 1975), Lecithochirium musculus (FILIPPI et al., 2012), and Schistosoma mansoni (HOCKLEY, 1973).In the present study, spines surrounding the body and suckers were of different shapes within parasite species, being either tongue-or claw-shaped, which agrees with the study of Lee et al. (1985Lee et al. ( , 1987) ) who concluded that the tegument spines have shapes and distributions that vary between parasite species, and these are related to the size or shape of worms and their migration pattern.This difference may also involve a significant difference in host-parasite relationships (CHAI et al., 2000).Køie (1977) suggested that large, pointed spines may be involved in feeding, with spines acting as abrasive structures for the host tissue.In contrast, Senft et al. (1961) and Lumsden (1975) opined that spines aided in the attachment to the host tissue.Several authors have also argued that spines could be involved in locomotion (BENNETT, 1975;URSONE & FRIED, 1995).The decrease in spine density on the non-feeding sucker (the oral sucker) may be related to a specialization of the area which facilitates attachment to the host tissue without causing damage (PANDEY & TEWARI, 1984).As in the present study, Dezfuli et al. (1997) also observed a decrease in spine density in   v. 27, n. 3, july.-sept. 2018 325/326 325 Urotrema scabridum and Renschetrema indicum: light microscopy and surface topography the ventral part of the small portion between the two suckers, while the lateral spines of this region are larger and stockier than any other spine observed on the entire body surface.The presence of domed papillae in the region of the oral sucker is frequently recorded in digeneans.The domed papillae are smooth, without apical cilia as in Calicophoron sp. and Erilepturus hamati reported in the studies of Tandon & Maitra (1982) and Abdou (2001), respectively; and not vesicular with a granular appearance as in Dicrocoelium dendriticum (CIFRIAN et al., 1988).These papillae may help in tactile and receptive functions (BENNETT, 1975;OTUBANJO, 1985).Both of the parasite species recovered in the present study showed a thick-lipped ventral sucker and papillae with intertwining folds; these folds and papillae help in creating a strong attachment to the host surface (TANDON & MAITRA, 1983).

Figure 2 .
Figure 2. Scanning electron micrograph showing the surface topography of Urotrema scabridum.(a) Whole ventral view.OS, oral sucker; VS, ventral sucker, Bar 5µm; (b) The terminal oral sucker with slit like mouth (MO) surrounded by papillae (arrow), Bar 20µm; (c) type I dome like papillae (arrows) surrounding mouth, Bar 10µm; (d) ventral sucker, the lip of this sucker was wrinkled and covered with tegument spines and few scattered papillae (arrows), magnified at the inset (arrows), Bar 10µm; (e) Tongue-shaped tegument spines below the level of the oral sucker, Bar 10µm; (f ) Tegument between oral sucker and the ventral pit covered with pointed tip spines, Bar 5µm.

Figure 4 .
Figure 4. Scanning electron micrograph showing the surface topography of Renschetrema indicum.(a) Whole ventral view; OS, oral sucker; MO, mouth opening; VS, ventral sucker; R, cytoplasmic ridges, Bar 20µm; (b) The anterior half of the worm, oral sucker includes the mouth opening which is directed anterioventrally and surrounded by papillae, the ventral sucker is pre-equatorial, VP ventral pit; S tegument spines, Bar 5µm; (c) Terminal portion of the body showing the oral sucker with two circles of papillae (arrows) surrounding the mouth opening, Bar 10µm; (d) Tegument ridges equipped with rows of spines with pointed tip, Bar 10µm; (e) The posterior extremity, devoid of spines, with the sub-terminal excretory pore (EP), Bar 10µm.

Table 1 .
Comparative measurements between U. scabridum and some species of the same genus recorded previously from different hosts.