Entobdella hippoglossi ” ( Müller, 1776 ) Blainville, 1818 (Muller, 1776)

Kearn, Graham C., Whittington, Ian D. & Evans-Gowing, Richard, 2007, A revision of Entobdella Blainville in Lamarck, 1818, with special emphasis on the nominal (type) species “ Entobdella hippoglossi (Müller, 1776) Blainville, 1818 ” (Monogenea: Capsalidae: Entobdellinae) from teleost flatfishes, with descriptions of three new species and a new genus, Zootaxa 1659, pp. 1-54 : 8-15

“ Entobdella hippoglossi ” ( Müller, 1776) Blainville, 1818

( Figs. 1–18, 32–34, 39–40)

1) Seminal receptacles. In spite of the large size of some stained and mounted adult specimens of “ E. hippoglossi ” from Atlantic halibut, H. hippoglossus (the largest preserved parasite specimen in our collection measured 2.25 cm in total length), the seminal receptacles were visible. In 3 adults, 4 seminal receptacles were identified in each specimen, with a possible fifth receptacle in 2 specimens. However, an immature specimen measuring 4.78 mm in total length (anterior hamulus length about 500) had a cluster of 9 seminal receptacles. At least 5 seminal receptacles arranged in a cluster around the ovo-vitelline duct were seen in wax-embedded facial sections of an adult specimen ( Fig. 1).

Six, possibly 7, seminal receptacles were arranged in a cluster around the ovo-vitelline duct ( Fig. 2) in resin-embedded facial sections of “ E. hippoglossi ” from Pacific halibut, Hippoglossus stenolepis . Seminal receptacles were visible in unstained whole mounts, but it was difficult to determine their number. Four receptacles were identified in 1 whole mount and at least 7 in another.

2). Vagina. The common genital opening was located on the left margin of the body, level with the posterior region of the pharynx in all entobdellines we examined. The external opening of the vagina, when visible, was widely separated from the common genital opening and located posterior and median to it, on the ventral surface left of the midline, as illustrated by Kearn (1970, text-fig.1) in E. soleae from the common sole, Solea solea (Linnaeus, 1758) ( Pleuronectiformes : Soleidae ) and by Klassen et al. (1989, figs. 1, 5) in “ E. hippoglossi ” from H. stenolepis .

For most of its length, the vagina in parasites from H. hippoglossus and H. stenolepis consists of a tightly convoluted tube arranged in a narrow column travelling across the body in a diagonal direction, roughly parallel to the female reproductive tract (oviduct, ootype, short uterus) ( Figs. 3 and 4 respectively). In parasites from H. stenolepis the vagina is illustrated by Klassen et al. (1989, figs. 1, 5) as a spirally coiled tube, but in our experience the convolutions are not as regular as those in their illustrations. The tube often contains fibrous material, presumably stored sperm, and in resin sections it has a thin wall and stains lightly with toluidine blue. Wax-embedded facial sections of “ E. hippoglossi ” collected from H. hippoglossus and resin sections of an adult specimen of “ E. hippoglossi ” from H. stenolepis , revealed that the main convoluted vaginal tube opens proximally into the vitelline reservoir ( Figs. 3 and 4 respectively), not into a seminal receptacle as stated by Klassen et al. (1989).

The illustrations of Klassen et al. (1989, figs. 1, 5), said to be composite drawings of parasites taken from H. stenolepis and Eopsetta jordani , show the spirally coiled vagina running anterolaterally from the seminal receptacle and opening on the ventral surface. However, we have found that the main, spacious vaginal tube of “ E. hippoglossi ” from H. hippoglossus and H. stenolepis does not extend as far as the vaginal opening but communicates with this opening via a short specialised region of the vaginal tract. Particularly surprising is the discovery of significant differences in the anatomy of this distal (terminal) region of the vagina among parasites collected from H. hippoglossus and previously assumed to be Entobdella hippoglossi .

In a few small, immature, unstained and cleared specimens of “ E. hippoglossi ” from H. stenolepis observed with phase contrast optics, it was possible to identify the vaginal opening and trace the path of the vagina into the parasite ( Fig. 5). The vaginal opening of 1 of these immature parasites measuring 5780 in total length was only 4 in diameter, giving access to a straight or slightly undulating thin-walled entrance tube about 60 long. The diameter of the entrance tube ranged between 4 and 6. This tube joined another short tube that followed a winding path and possessed a relatively thick, possibly glandular, wall ( Fig. 5). This tube was about 50 long and gave access proximally, via a restricted communication, to the main vagina, the diameter of which rapidly widened to about 16. This arrangement of the distal region of the vagina was confirmed in semi-thin resin sections of an adult parasite from H. stenolepis ( Fig. 6). The wall of the narrow, winding tube linking the narrow entrance tube and the wide main vagina, stained with toluidine blue ( Fig. 6).

A preliminary survey of a range of specimens of “ Entobdella hippoglossi ” from H. hippoglossus revealed remarkable differences in vaginal anatomy. The vaginal opening was not found in a sample of 8 parasites (3 adults and 5 immature), hereinafter referred to as “ E. hippoglossi type 1”, supplied by Dr A. Shinn from the skin of H. hippoglossus ( Table 1), in spite of an intensive search using a compound microscope in the region of the ventral surface where it would be expected to be. However in 4 immature specimens, the vaginal tract was identified running diagonally posteromedianly from the ventral region where the vaginal opening is assumed to lie. No anatomical differentiation of the distal region of the vagina could be resolved in these specimens. However, in toluidine-blue-stained resin sections of an adult parasite from this sample, the vaginal opening was located ( Fig. 7). This opening was so small (diameter in Fig. 7 about 2) that failure to locate it in whole mounts is not surprising. The distal terminal region of the vagina was similar to that of “ E. hippoglossi ” from H. stenolepis . The opening gave access to a thin-walled, relatively straight entrance tube only 6 – 7 in diameter and about 85 long ( Fig. 8). Between this thin-walled entrance tube and the first coil of the spacious main vagina was a short tube that followed a winding path, with a lumen even narrower than that of the entrance tube and a wall with a strong affinity for toluidine blue ( Fig. 8). This stained region, measured in a straight line from the proximal end of the entrance tube to the first coil of the main vagina, is about 65 long, so that the whole of this modified distal region is short (about 150 long) compared with the coiled vaginal tube, which, measured in a straight line, covers a distance well over 1 mm in an adult parasite of moderate size (1.1 cm long).

In a second group of parasites from H. hippoglossus from various sources, hereinafter referred to as “ E. hippoglossi type 2”, the vaginal opening was conspicuous and usually easy to locate in cleared and mounted parasites ( Fig. 40A). Two features contributed to this conspicuousness: (a) the opening in these individuals was relatively large (diameter approximately 50) ( Fig. 40A) and gave access to an atrium just beneath the ventral surface ( Figs. 9, 10, 12); (b) the vaginal opening and atrium were surrounded by a circular array of densely packed concentric fibres situated below the ventral tegument ( Figs. 12, 40A). Similar ventral fibres, arranged concentrically around the vaginal opening but not as densely packed, have been detected in E. soleae (see Kearn 2006). In 3 of the “ E. hippoglossi type 2” specimens, assumed to be from H. hippoglossus from the Aberdeen Fish Market ( Table 1) and measuring 1.5, 1.9 and 2.0 cm in total length, the external diameter of the circular array of fibres measured approximately 360, 440 and 320, respectively. Frequently the vitelline follicles were displaced by the vaginal atrium and associated tissue, adding to the prominence of this anatomical feature.

Adult specimens of “ type 2” tend to be larger (up to 2.25 cm in total length) than specimens of “ type 1”, although there is considerable overlap in size between the 2 types in our collection of parasite specimens. The largest “ type 1” specimen was 1.6 cm long ( Table 1; unknown source from Faroe Bank) and the smallest “ type 2” specimen was 0.67 cm long ( Table 1; MZUB no. 47982 from East Greenland) .

In sections of a resin-embedded specimen of “ E. hippoglossi type 2” with a conspicuous distal vagina, the surface opening of the vagina was funnel-shaped and narrowed briefly before expanding slightly to form the atrium ( Fig. 9). The atrium lining stained intensely with toluidine blue, as did the fibres (assumed to be muscle) associated with the vaginal opening. The atrium communicated proximally with the first coil of the main vaginal tube by way of a constricted opening ( Figs. 10, 11). The first few coils of the vagina travelled ventrally ( Figs. 9, 10, 11). The remaining coils were arranged in a straight narrow column following a posteromedian course across the body.

Thus, with regard to the anatomy of the distal region of the vagina, specimens of “ E. hippoglossi ” from H. hippoglossus fall into 1 of 2 categories, designated “ E. hippoglossi type 1” (lacking a vaginal atrium; vaginal opening small and inconspicuous and not surrounded by a compact ring of superficial fibres) ( Fig. 32A) and

“ E. hippoglossi type 2” (with a conspicuous vaginal atrium and a large and conspicuous vaginal opening surrounded by a compact ring of superficial fibres) ( Fig. 40A).

The ventral surfaces of “ E. hippoglossi type 1” from H. hippoglossus and “ E. hippoglossi ” from H. stenolepis were searched for sub-tegumental fibres arranged in a circular pattern around the vaginal opening or around the location where the vaginal opening was judged to be. No circular fibres were found in “ E. hippoglossi type 1”, but in 1 specimen from H. stenolepis a circular pattern of sub-tegumental fibres was observed ( Fig. 39A). These fibres did not form a compact ring immediately surrounding the vaginal opening as in “ E. hippoglossi type 2”, but were rather remote from it ( Fig. 39A). A partial ring of fibres in a similar location was found in a second specimen from H. stenolepis .

3). Anterior adhesive pads. All parasites examined in this study had 2 adhesive pads, 1 on each side of the head. Using SEM, Kearn & Evans-Gowing (1998, fig. 3) found that each pad of E. soleae is divided by 2 narrow transverse strips of body tegument into 3 separate adhesive sub-units. Similarly the adhesive pads of “ E. hippoglossi types 1 and 2” from H. hippoglossus and “ E. hippoglossi ” from H. stenolepis are divided into 3 sub-units but they are not always easy to see. It is sometimes necessary to resort to SEM to resolve this arrangement, as in Fig. 13 (“ E. hippoglossi type 2”) and Fig. 14 (“ E. hippoglossi ” from H. stenolepis ), but in other specimens the sub-divisions can be distinguished using reflected light.

4). Haptor. The accessory sclerites of “ E. hippoglossi types 1 and 2” from H. hippoglossus and “ E. hippoglossi ” from H. stenolepis are slightly curved when viewed in the appropriate orientation ( Fig. 15A, B, C), but appear virtually straight when the viewpoint is rotated 90º. The distal half of the sclerite is spear-shaped, the tapering distal region protruding from the ventral surface of the haptor. In some particularly large specimens of “ E. hippoglossi type 2”, the pointed distal tips of the accessory sclerites are missing ( Fig. 15B). In addition, the anterior (proximal) regions of the anterior hamuli of some large individuals of both “ E. hippoglossi type 1” and “ E. hippoglossi type 2” are distorted, suggesting that these regions of the sclerites have lost their rigidity. Tendons associated with the accessory sclerites of “ E. hippoglossi type 1” ( Fig. 32A), “ E. hippoglossi type 2” ( Fig. 40A) and “ E. hippoglossi ” from H. stenolepis ( Fig. 39A) terminate at the anterior end of the anterior hamuli.

The ratios of the lengths of the anterior hamuli and accessory sclerites for samples of “ E. hippoglossi types 1 and 2” from H. hippoglossus and for a sample of “ E. hippoglossi ” from H. stenolepis are given in Table 3. The mean ratios for “ E. hippoglossi ”, irrespective of source and body size, were rarely more than 2. In 2 immature specimens of “ E. hippoglossi type 1” the mean sclerite ratio was 1.45 and a mean based on 10 adults was 1.66 ( Table 3). In 3 immature specimens of “ E. hippoglossi ” from H. stenolepis , the mean sclerite ratio was 1.7 and a mean based on 10 adult specimens was 1.71 ( Table 3).

Papillae are present on the ventral haptor surfaces of “ E. hippoglossi type 1” ( Fig. 16), “ E. hippoglossi type 2” ( Fig. 17) and “ E. hippoglossi ” from H. stenolepis ( Fig. 18). Lyons (1973) described similar papillae in E. soleae (see below) and found evidence to suggest that they may have a sensory function, possibly as contact or strain receptors. The size, shape, number and distribution of papillae provided a useful and practical means of distinguishing “ E. hippoglossi type 1” from “ E. hippoglossi type 2” (cf. Fig.16 with Fig. 17 and Fig. 32A with Fig. 40A). The number and distribution of papillae on the ventral surfaces of the haptors of 23 unflattened, unmounted and uncleared parasites collected from a mature female H. hippoglossus (total length 178 cm) from the Faroe Bank ( Table 1) were compared using reflected light. This revealed 11 specimens of “ E. hippoglossi type 1” and 12 “ E. hippoglossi type 2”.

“ Entobdella hippoglossi type 1” has fewer haptor papillae than “ E. hippoglossi type 2”. The mean densities were 45 (33 – 59) papillae per mm 2 (n = 8) and 129 (105 – 156) papillae per mm 2 (n = 7), respectively. Papillae density in a single adult “ type 1” and 1 adult “ type 2” specimen both with the same total body length (1.4 cm), was 39 papillae per mm 2 and 124 papillae per mm 2, respectively. It also appears that the papillae of “ E. hippoglossi type 1” are larger than those of “ E. hippoglossi type 2”. However, the density of papillae (especially in “ E. hippoglossi type 2”) and regional differences in papillae size made objective measurements difficult. The diameters of the largest and smallest papillae for the 2 “ types ” ranged between 30 and 125 for “ E. hippoglossi type 1” and 20 and 95 for “ E. hippoglossi type 2”. Thus papillae of “ E. hippoglossi type 1” regularly exceed 100 in diameter while papillae of “ E. hippoglossi type 2” do not.

The haptor papillae of “ E. hippoglossi type 1” possess fleshy apical protuberances. These protuberances are often bilobed ( Fig. 16 and inset) and papillae with 3 or 4 apical lobes are occasionally encountered. Apical protuberances are visible in immature specimens of “ E. hippoglossi type 1”. The papillae of “ E. hippoglossi type 2” are roughly circular ( Fig. 17) and appear to lack apical lobes. Some SEM photographs showed that the papillae of “ E. hippoglossi type 1” have circular ridges; these were not observed on papillae of “ E. hippoglossi type 2” (cf. Figs. 16, 17) .

* Two ratios (2.03 and 2.05) exceeded 2.0.

**Calculated from Brinkmann (1952a).

The papillae occupying the lateral fields of the haptor (lateral to the median sclerites) are arranged in conspicuous radial rows in “ E. hippoglossi type 1” ( Fig. 32A). In “ E. hippoglossi type 2”, the papillae in the corresponding regions of the haptor are so numerous and dense that any such arrangement is obscured ( Fig. 40A). However, in some specimens a radial arrangement of some of the lateral papillae can be discerned. The papillae of “ E. hippoglossi type 2” extend further in an anterior direction than those of “ E. hippoglossi type 1” (cf. Fig. 32A with Fig. 40A). In most specimens of “ E. hippoglossi type 2”, papillae lie anterior to the region where the peduncular connection from the body joins the haptor ( Fig. 40A). In “ E. hippoglossi type 1” the papillae do not extend anteriorly beyond the posterior margin of the peduncle ( Fig. 32A).

The papillae of “ E. hippoglossi ” from H. stenolepis tend to lie in radial rows in the lateral regions of the haptor ( Figs. 18, 39A) and have a density of 75 (59 – 97) papillae per mm 2 (n = 5). The diameter of the papillae ranged between 30 and 70. SEM shows that the papillae lack circular ridges and fleshy apical protuberances like those found in “ E. hippoglossi type 1” (see above) .

5). Other features. In 3 specimens of “ E. hippoglossi type 1” from H. hippoglossus and in 5 specimens of “ E. hippoglossi ” from H. stenolepis the 4 pigment cups of the eyes were conspicuous (cf. Fig. 32A with Fig. 39A). The eyes were not found in 6 out of 8 parasite specimens of “ E. hippoglossi type 2” from, or assumed to be from, H. hippoglossus ( Fig. 40A). In 1 of the other 2 “ type 2” parasite specimens, the 2 anterior eyes were absent and in the other, the posterior eyes were present but hard to see.

Glands of Goto are present in the posterior angle between the testes in “ E. hippoglossi types 1 and 2” from H. hippoglossus and in “ E. hippoglossi ” from H. stenolepis (see Figs. 32A, 40A, 39A respectively).