Additional morphological characters of the nematode Capillostrongyloides fritschi (Travassos, 1914) Moravec, 1982 (Nematoda: Capillaridae) from Bagrus spp. (Bagridae) inhabiting Damietta Branch of the River Nile, Egypt with a special reference to evaginated cirrus

The catfish Bagrus bajad (Forsskål) and B. docmak (Forsskål) (Siluriformes: Bagridae) are well distributed in the River Nile and have an economic value with good marketing and use in aquaculture. Collected specimens of Capillostrongyloides fritschi (Travassos 1914) were redescribed using a phase-contrast microscope and scanning electron microscope. The most remarkable finding was the unique structure of the evaginated cirrus, being composed of a proximal long tube, a middle spherical bulb packed with mature spermatozoa, and a distal funnel-shaped structure. All regions of the cirrus are provided with longitudinal and transverse muscles and covered with transverse cuticular folds. The mechanism of cirrus emergence was discussed based on available musculature data. Other important findings are the short stylet of the mouth, the two lateral oral lobes, 6-10 buried cephalic papillae and the bacillary bands that lack elevations. Stichosome is formed of 35-44 stichocytes that were differentiated into black and white forms, each containing dense granules and translucent vacuoles. The intestine showed a convoluted part at its anterior region. The vulvar lips are slightly elevated, many mature eggs are cleaved, and their shells are 3-layered. The anus was found in a groove bounded by two unequal lobes.


Introduction
The River Nile freshwater fish are important animal protein sources (Samy-Kamal, 2015). The catfish Bagrus bajad (Forsskål) and B. docmak (Forsskål) (Siluriformes: Bagridae) are well distributed in the River Nile and have an economic value with good marketing and use in aquaculture (Mashaly et al., 2019). In a recent ecological study of helminth parasites of Bagrus spp. from the Damietta Branch of the River Nile, Allam et al. (2023) recorded a nematode species, Capillostrongyloides fritschi (Travassos, 1914) Moravec, 1982 (Nematoda: Capillaridae) from the stomach and intestine. Moravec (2001) considered the genus Capillostrongyloides as one of the allegedly valid genera in the family Capillariidae. The genus currently includes 10 valid species, namely, C. fritschi, C. sentinosa Travassos, 1927, C. tandani Johnston & Mawson, 1940C. physiculi Johnston & Mawson, 1945, C. tasmanica a stichosome with a single row of stichocytes, the presence of a well-developed bursa supported by two short and broad lateral lobes, each of them bearing a papilla, and a non-spiny spicular sheath (Moravec, 2001). In Egypt, C. fritschi was described by Tadros & Mahmoud (1968) as Capillaria yamagutii from the stomach of B. bajad, and B. docmak. Imam et al. (1991) reported the same species from the same host and locality while Moravec (1974Moravec ( , 2001 revised the same nematode species and considered it a synonym of C. fritschi. Previous descriptions of C. fritschi are incomplete since they have been concentrated on the morphometrics and little is known of the structural details of the cephalic region, digestive system, and reproductive system of both females and males, particularly the cirrus (spicular sheath) and vulva. The present study aims to redescribe the capillariide C. fritschi using the phase-contrast microscope and scanning electron microscopy. Special attention has been given to the cirrus, in invaginated and evaginated positions since they are highly used for the differentiation of the congeneric species.

Materials and Methods
A total of 141 individuals of the freshwater catfishes Bagrus bajad and Bagrus docmak were collected from the Damietta branch of the River Nile at Mansoura city from March 2020 to February 2021. Living captured fish were transported in an aerated container, with fresh water, to the parasitology laboratory at Mansoura University and kept alive in an aquarium with aerated water. The parasites were collected and examined following the method of Allam et al. (2023) while their identification and measurements were done according to Moravec (2001). All measurements and scale bars were calculated on 20 living adults using OMAX TopView 3.7 program. For scanning electron microscopy (SEM), 10 living adults were preserved, postfixed, and dehydrated according to El-Naggar et al., (2016). Critical point drying was performed using EMS Q 850 critical point dryer with carbon dioxide as the transition fluid. The specimens were then coated with gold using SPI Sputter Coater and examined with a JEOL JSM 6510 lv electron microscope at the Electron Microscope Unit (EMU) of Mansoura University.
Posterior body region provided with well-developed transverse muscle bands, particularly at middle region of spicule (Fig. 3d). Caudal end rounded and provided with well-developed membranous bursa supported by 2 wide, ventrolateral lobes (Figs. 1b,2c,d,3a). One relatively large papilla present at base of each lobe (Fig. 2 c, d). Surface of ventrolateral lobes and Cephalic region (frontal view). b. Cephalic region (lateral view). c. Caudal end (ventral view). d. Caudal end (Dorsal view). e. Caudal end (evaginated spicular sheath). Scale bar: a and d = 2 lm; b = 1 lm; c = 3 lm; e = 5lm. Abbreviations: ac, anterior concavity; co, cloacal opening; con, constriction between long cylindrical tube and spherical bulb; cr, cephalic region; cts, cuticular transverse striations; dt, distal wider region; icf, irregular cuticular folds; le, longitudinal elevations; m, mouth opening; mb, membranous bursa; p, pore; pc, posterior concavity; pt, proximal region of long cirrus tube; rcf, radial cuticular folds; sb, spherical bulb; stl, finger-like stylet; tcf, transverse cuticular folds; ti, transverse incision; vll, ventrolateral lobe; vlp, ventrolateral papilla; small white arrow, buried cephalic papilla; large white arrow, oral lobe. membranous bursa covered with corrugated cuticle (Fig. 2c, d). In living male specimens, evaginated (everted) spicular sheath (cirrus detected in 4 sequent patterns of emergence (Figs. 1c-f, 2e, 3a-d). In first pattern, cirrus emerging as long, cylindrical tube supported by longitudinal muscle and ending with small bulb provided with transverse circumferential muscles (Figs. 1c,3a). In second pattern, cirrus consisting of long cylindrical tube (proximal region) containing terminal part of spicule and ejaculatory  SEM showed that the everted cirrus is not completely evaginated and only the long cylindrical tube and spherical bulb are seen emerging from the cloacal opening (Fig. 2e). None of the short tube and funnellike regions were seen in this specimen. The basal part of the tube is relatively narrow and formed of parallel longitudinal elevations that probably represent underneath longitudinal muscles (Fig. 2e). The distal part of the tube, which is wider than the basal one, is covered by transverse cuticular folds and shows two circular, smooth, concavity-like structures (anterior and posterior) (Fig. 2e). The bulb region, which is wider than the cylindrical tube, is covered with considerably large, radially orientated cuticular folds and terminates with a single median pore (Fig. 2e).

Discussion
A redescription has been done for the nematode C. fritschi infecting the intestine of the catfish B. bajad and B. docmak (Bagridae) from the Damietta branch of the River Nile. This is the first time to describe this nematode using Scanning Electron Microscopy (SEM) and provide more additional morphological data of the cephalic and caudal regions, digestive system and reproductive system including invaginated and evaginated cirrus and vulva. The present morphometrical data of C. fritschi resemble, in general, those reported by Moravec (2001) (see Table 1    Abbreviations: ann, annuli; cit, convoluted intestine; dg, dense granule; dst, dark stichocyte; egs, eggshell; go, glandular oesophagus; il, inner thin layer; lbl, large blastomere; lst, light stichocyte; meg, mature egg; ml, middle hyaline layer; mo, muscular oesophagus; N, nucleus; nit, narrow intestine; nr, nerve ring; ol, outer thick layer; sbl, small blastomere; st, stichosome; stl, finger-like stylet; tv, translucent vesicles; ut, uterus; va, vagina; vam, vaginal muscles; vu, vulva; vul, vulvar lip; wit, widened region of intestine; white arrow, two polar plugs; black arrows, wing-like cells. differentiated into 3 layers; 2 layers by Moravec (2001).
Most species of Capillostrongyloides exhibit a rather uniform morphology and are mainly differentiated by the body length, the length of spicules, the size and shape of the evaginated spicular sheath (cirrus), the shape and length of the caudal lobes and membranous bursa, and presence/absence of elevated vulvar lips. Few of the previous studies on Capillostrongyloides species (Moravec, 2001;González-Solís et al., 2014) have described the male copulatory organ (cirrus) in the evaginated state. In the present study, the cirrus could be detected in successive stages (patterns) of evagination, and the structure of a completely everted cirrus is described in detail using phase contrast and incompletely everted by SEM. The completely evaginated cirrus consists of three main regions: the proximal long tube, the middle spherical bulb, and the distal funnel-shaped structure. Part of the terminal portion of the ejaculatory duct and spicule extend inside the proximal region where the ejaculatory duct dilates into a spacious cavity packed with mature spermatozoa in the bulb region. In the original description of C. fritschi made by Travassos (1914)  Cephalic region (ventral view).. b. Bacillary bands (lateral view). c. Vulva (ventral view). d. Caudal body region (ventrolateral view). Scale bars: a = 2 lm, b,c and d = 5 lm. Abbreviations: an, anus; avl, anterior vulvar lip; ccf, corrugated cuticular folds; cr, cephalic region; cts, cuticular transverse striations; m, mouth; pl, posterior lobe; pvl, posterior vulvar lip; stl, finger-like stylet; ti, transverse incision; vo, vulvar opening; small white arrow, circular openings of underneath bacillary cells; small black arrow, buried cephalic papillae; large white arrow, long groove. present study, the evaginated cirrus of C. fritschi is unique among other described Capillostrongyloides species or indeed among other species belonging to the family Capillaridae. Among 10 previously described species of the genus Capillostrongyloides, only two species were reported to show evaginated cirrus, C. ancistri (see Moravec, 1987), and C. morae (see González-Solís et al., 2014). In both species, the evaginated cirrus is a cylindrical tube without a bulb or funnel-shaped structure that is present in the cirrus of C. fritschi. Variations have been reported in the structure of the cirrus in other members of the family Capillaridae. In Paracapillaroides acanthocotylus Rossin & Timi, 2009, evaginated cirrus consists of a short, cup-shaped, spinose, proximal part and a long, narrower, tubular, non-spiny distal part, indicating the absence of the spherical bulb and the funnel-shaped distal region. The evaginated cirrus is an ampullashaped structure in Amphibiocapillaria combesi Chabaud & Knoepffleri, 1985, a very long cylindrical tube with protruding spicule in Capillaria (Capilloroides) catenate Van Cleave & Mueller, 1932, Amphibiocapillaria tritoniscnstati (Diesing, 1861) Moravec, 1982, andA. tritonispunctati (Diesing, 1851) Moravec, 1982, a very long tubular structure without spicule in Paracapillaria (Ophidiocapillaria) cesarpintoi (Freitas & Lent, 1931) Moravec, 1986, a very long tube with a globular expansion at its proximal end in Lobocapillaria austropacifica (see Moravec & Beveridge, 2017) or a bell-shaped and relatively short cylindrical tube with the spicule projecting from its opening in Paratrichosoma recurvum (Solger, 1877) Ashford & Muller, 1978. As in the present study, most of the previously described evaginated cirri are covered with transverse cuticular folds (e.g., Pseudocapillaria (Pseudocapillaria) carangi (Parukhin, 1971) Moravec, 1982. The mechanism of copulation has not been observed in living C. fritschi or in other capillarids. Most male nematodes with spicules at their posterior region are used to spread and open the vulva and vagina during copulation, with the aid of the other accessory reproductive structures (Wharton, 1986). In male nematodes which lack spicules as in the Table 1 Comparison between measurements (in lm) of the present study and those of Moravec (2001) of the nematode, Capillostrongyloides fritschi.

Female
Present study Moravec, 2001 Male Present study Moravec, 2001 Total  (Fürst von Lieven et al., 2005). In the present study, most of the whole length of the spicule remains inside the body with only a short part of its distal region protruding with the evaginated cirrus. The everted cirrus of C. fritschi, is supported by transverse cuticular folds at the proximal and middle regions as well as fine and thick transverse and longitudinal muscle fibers along its length. It seems possible that these cuticular folds serve to keep the rigidity of the everted cirrus and prevent it from being collapsed during copulation. Moreover, contraction of the transverse muscles and relaxation of the longitudinal muscles may serve to elongate the cirrus and enable the funnel-shaped terminal end to enclose the lips of the vulva and make a firm attachment. Meanwhile, contraction of the transverse muscles around the bulb and relaxation of the sphincter muscles of the tube connecting the bulb with the funnel can create great pressure on the bulb cavity and leads to the movement of the mature spermatozoa into the funnel and subsequently to the vulvar opening. Contraction of the longitudinal muscles and relaxation of the transverse muscles can lead to the shortening of the cirrus and withdrawal of the funnel. The posterior body region of C. fritschi is supported by accessory genital organs like the membranous bursa, the ventrolateral lobes and posteriorly directed papillae. The real functions of these organs are not known but seem likely to secure a firm attachment of the male posterior region to the female body, thus facilitating the emergence of the cirrus during copulation. Also, contraction of the transverse circumferential muscles encircling the posterior body region may assist in the projection of the cirrus from the cloacal opening as the first step in copulation. In all these steps, a small part of the spicule was seen in the proximal region of the cirrus indicating that the spicule moves outside the cloacal opening and contributes to the emergence mechanism of the cirrus but has no role in widening the vulva during copulation as suggested for spicules in many other nematodes (Fürst von Lieven et al., 2005). For a concise understanding of the copulation process, living males and females might be examined while copulating using a stereomicroscope and SEM. Moreover, the musculature of the copulatory organs in both males and females should be studied in detail with the fluorescent and confocal laser scanning microscope to show the different types of muscles involved in the copulation process.
In the original description of C. fritschi made by Travassos (1914) and the description made by Moravec (2001), the bacillary bands are covered by tiny papillae-like formations and the mouth has no stylet while in the present study, the stylet is present, but papillae-like formations have not been detected either with the light microscope or SEM. The stylet was not described in all adult species of the genus Capillostrongyloides (see Santos et al., 2008 andCantatore et al., 2009) except C. ancistri (see Moravec, 1987) and C. morae (see González-Solís et al., 2014). However, the first-stage larvae of Trichuris trichiura Linnaeus, 1771, Trichinella spiralis Owen, 1835 and Trichosomoides crassicauda (Bellingham) were shown to have distinct stylets, a feature which led Anderson (1992) to believe that the presence of a stylet in the first-stage larva is characteristic of all trichinelloids. In capillariids, Wright (1974) observed the stylet in the buccal cavity of adult Calodium hepaticum Bancroft, 1893 and suggested that the stylet can probably remain without projection from the mouth (Wright, 1961). However, a functional stylet was observed by Baruš et al. (1981) in adult Capillaria anatis Schrank, 1790 and by Moravec & Justine (2014) in Capillaria plectropomi. The finding of functional stylets in the previously mentioned capillariids suggests its presence in all adult capillariids (Moravec & Justine, 2014). The stylets in adult nematodes may serve in the withdrawal of food and could share in the metabolic process (Moravec, 2001).
The cephalic region of C. fritschi closely resembles that of C. morae described by González-Solís et al. (2014), C. anatis Schrank, 1790 by Baruš et al. (1981), Paracapillaria philippinensis Chitwood, Velasquez & Salazar, 1968 by Moravec (2001), C. plectropomi by Moravec & Justine (2014), Capillaria appendigera n. sp. (see Moravec & Barton, 2018), Paracapillaria gastrica n. sp. (see Moravec & Justine 2020), except for the amphids and oral lips which are not clearly visible and the cephalic papillae which are lesser in number and buried in the cuticular surface. In these capillariids, 12 cephalic papillae (sessile or buried) were observed (distributed in two circlets) on the cephalic plate around the oral aperture. Regarding their position in the cephalic region, these papillae may act as chemoreceptors.