A total of 6 hippopotami (Hippopotamus amphibius: 4 males and 2 females) were examined in Mpumalanga Province, South Africa in August and September 2016. The geographical coordinates for the hippopotamus examined are listed in Table 1. After collecting monogeneans, the host tissue was biopsied and fixed with 10% buffered formalin for histopathological studies. For the morphological examination, parasites were relaxed in a small Petri-dish with 0.9% saline, and while between 2 microscope slides were fixed by 70% ethanol.

Worms were stained in Mayer’s acid carmine, destained in 4% hydrochloric acid in 70% ethanol, dehydrated in ascending concentrations of ethanol (12 hr each), and cleared in 100% xylene and then in 50% Canada balsam and 50% xylene (12 hr each). Whole worms were then mounted in Canada balsam.

Specimens fixed and stored in 70% ethanol were processed following standard methods [11], which included critical point drying in sample baskets and mounted on SEM sample mounts (stubs) using conductive double-sided carbon tape. Samples were coated with gold and palladium for 3 min using a Polaron #3500 sputter coater (Quorum (Q150 TES) www.quorumtech.com) establishing an approximate thickness of 20 nm. Samples were placed and observed in an FEI Helios Dual Beam Nanolab 600 (FEI, Hillsboro, Oregon, USA) Scanning Electron Microscope and digital images were obtained in the Nanolab software system (GEI, Hillsboro, Oregon, USA). Samples were viewed under low vacuum conditions using 10 kV, spot size 2, 0.7 Torr using a GSE detector.

Standard methods, similar to the SEM procedure, were used for preparation. Specimens were examined and positioned with the above SEM instrument, which was equipped with a Phoenix energy-dispersive x-ray analyzer (FEI). X-ray spot analysis and live scan analysis were performed at 16 kV with a spot size of 5 and results were recorded on charts and stored with digital imaging software attached to a computer. The TEAM (Texture and Elemental Analytical Microscopy), a modification of the EDXA system (Energy Dispersive X-ray analysis), software system (FEI) was used. The data included the weight and atom percentages of the detected elements following correction factors.

Biopsied host tissues with attached monogeneans previously fixed with 10% formalin were processed by the standard methods for paraffin-blocked specimens [12,13]. The paraffin-blocked tissue was sectioned at 4–5 microns, placed on glass slides and stained with hematoxylin and eosin (H&E). Additional sections were stained with Mallory’s trichrome to study the pathological responses to the parasite [14]. H&E as a standard stain for tissue whereas Mallory’s trichrome to differentiate granular tissue typical of parasite invasion. The prepared sections were viewed with a LSM laser (Carl Zeiss, Thornwood, New York, USA) equipped compound microscope and representative pictures were taken at varying magnifications with a digital camera. The histopathological sections were selected from a much larger collection of sections on 63 glass slides in RAH’s collection. The following abbreviations were used in histopathological studies: CF–collagenous fibers, Ep–stratified epithelium, H–hemorrhaging, N–necrosis, S–sucker.

A total 5 out of 6 examined hippopotami (83.3%) were infected with O. hippopotami, intensity range 4–97 (36) (Table 1). Commonly both eyes of hippopotami were infected (Table 1). Parasites had a firm grip on host tissues and they were never found detached. Most of these parasites were found on the nictitating membrane of eyes, and seldom on the side of the eye globe (Fig. 1). Different attachment sites were seen on the host’s eyes (Fig. 1C, D) with parasites forming clusters on several cases.

Distinguishing features between adults and sub-adults of O. hippopotami Monogeneans of 2 age groups, adults and sub-adults were recorded from the hosts. Two hosts were infected with adults only, while 1 host was uninfected (Table 1). Adults were easily distinguished from sub-adults with the naked eye: they were large, with a cherry-red anterior 2/3 of the body, collected in clusters of at least 5 or more individuals (Figs. 1, 2). Sub-adults were found separately from the cluster of adults and were noticeably smaller (Fig. 2), with an underdeveloped reproductive system. They were colorless, or white-yellowish to slightly pinkish (Fig. 1A). X-ray scans also showed major differences between the sub-adult and adult (see below). The most distinctive feature between the 2 age groups was the body size. Adults of O. hippopotami were on average 4.6 times larger than sub-adults (Fig. 2A, B; Table 2). Measurements of adults and sub-adults and comparisons with the original description and latest report [4] provided in Table 2. Sub-adults found in the present study varied in size and degree of development. The presence of a mixture of different generations on the same host for Oculotrema (in contrast to Polystoma Zeder, 1800) was a novel observation in the present study.

Measurements listed in Table 2. The average haptor width/haptor sucker ratio was 3.2 (Table 2) and the average body/haptor length ratio 6.5. The musculature of the oral sucker in the adult well developed, with few tissue layers (Fig. 3C). Ventral part of the body flattened, possibly due to the life-long mode of attachment of the worms. Likewise, the oral sucker opening also oriented in the same plane (Fig. 3B). The genital system is shown on Fig. 2C. The muscular cirrus with ejaculated sperms are shown in Figs. 3C, D. Arrows on Figs. 3C, E show the position of a genital papilla present in adults of O. hippopotami. Figs. 3F, G show the haptor and one of 6 well-developed suckers of an adult, designed for a very firm long-term attachment to the host tissue. The haptor width/haptor sucker ratio measured for the current study was 3.45 (3.62–3.67) in comparison with up to 4.5 of Du Preez et al. [4]. Tegument consists of plates elastically connected to each other and strands between plates. The attachment of sclerotized structures also differs structurally (see X-rays analysis part of the study). The external edges of suckers in the sub-adult form may be folded or corrugated (Figs. 4A, F, G).

Measurements listed in Table 2. The average haptor width/haptor sucker ratio was 2.9. The average body/haptor length ratio in sub-adults was 3.25. The musculature of the oral sucker undeveloped (Fig. 4B, C for a close view). Ventral part of the body flattened. Oral sucker opening oriented at an angle to the plane of the ventral part of the body (Fig. 4B). Genital papilla not seen. Suckers not as well developed as in the adult forms. Haptor width/haptor sucker ratio was 3.01 (2.50–3.52). Reproductive organs of the smallest sub-adults were not developed. The tegument consists of plates elastically connected to each other (Fig. 4D) with strands between plates. The tegument had a honeycomb-structure (Fig. 4E). En face view of the 6 muscular suckers of O. hippopotami depicted in Fig. 4F. The position of suckers and their orientation was similar to the adult form, but the relative ratio of the sucker to the haptor and haptor to body differed (Table 2). Haptor suckers were smaller and less developed in comparison with adults (Fig. 4G).

A lateral orbit of the eye socket of a hippopotamus was shown in Fig. 5A. This figure shows the surface of the eye of the host; a stratified epithelial lining with a connective tissue base below the basement membrane. There were extensive hemorrhaging (disruption of capillary vessels) and pooled blood cells as well as necrotic tissue in the area where parasites attached (Fig. 5B), which displayed a section of an infected hippopotamus eye pulled away from the surface of the eye. It displayed the 6 marks of host tissue where suckers attached. An area of the host eyes where specimens of O. hippopotami had been attached shown in Fig. 5C. Note the difference between normal host epithelium and epithelium in the invaded area (bottom left). The eye surface of the host has been altered as a result of the firm grip of parasites. Note tissue necrosis and hemorrhaging on the edges of the marking (Fig. 5D). See also Fig. 5E, showing host epithelial eye cells surrounding the point of attachment. Note the extensive encapsulation with collagenous fibers, in an attempt of the host to “wall-off” the parasite. Fig. 5F displayed the effect of a parasite sucker attaching to the host epithelium. Note the extensive tissue necrosis in the center of the attachment area of the sucker. Also note extensive necrosis caused the sucker and hemorrhaging of host tissue (Fig. 5G). The effect of parasite suckers’ attachment to the surface of the eye and the resulting necrotic tissue inside the sucker depicted in Fig. 5H. Note small capillaries scattered throughout the eye epithelium with red blood cells in the vessels. Compare this to Fig. 5I, which showed 3 of the 6 O. hippopotami suckers with a disruption of eye tissue and hemorrhaging with resulting cell necrosis. Note the corrugated nature of the sucker with specialized collagenous fibers. Compare to Fig. 5G with a single intact worm sucker surrounding host epithelial cells.

Table 3 and Figs. 6, 7 contained the data for the X-ray scans of the adult and sub-adult worms. Three areas were scanned, i.e., body, mouth and sucker. Duplicate scans were done on different specimens for a qualitative evaluation to understand changes in the chemistry of the organism during its lifespan. The processing and coating chemicals (Au, Pd and Os) were omitted from the table. Note the definite lack of hardening ions (Ca, S, P) in the scanned areas of the sub-adults. The common chemical ions (C, N and O) in living protoplasm were consistent for both life stages.