Introduction

Members of the genus Micropleura von Linstow, 1906 are dracunculoid nematodes of the family Micropleuridae found in the peritoneal and abdominal cavities of their crocodilian and chelonian hosts (Anderson, 2000). According to Siddiqi & Jairajpuri (1963), first-stage larvae released into the water by the viviparous females develop to the infective stage in Cyclops spp., but as yet little is known about the biology and life history of Micropleura spp.

The genus Micropleura was erected to accommodate M. vivipara, described from Gavialis gangeticus (Gmelin in L.) (Gavialidae) in India, and at present, a further three species are considered valid: M. vazi Travassos, 1933 from Caiman crocodilus (L.) (syn. Caiman sclerops Gray) (Alligatoridae) in Brazil (type-locality) and Venezuela, M. australiensis Moravec, Kay & Hobbs, 2004 from Crocodylus johnsoni Krefft (type-host) and Crocodylus porosus Schneider (Crocodylidae) in Australia, and M. indica Khera, 1951 from Nilssonia gangetica (Cuvier) (syn. Trionyx gangeticus Cuvier) (type-host) and Lissemys punctata (Lacépède) (Trionychidae) in India (von Linstow, 1906; Travassos, 1933a, b; Khera, 1951; Siddiqi & Jairajpuri, 1963; Moravec & Prouza, 2003; Moravec et al., 2004, 2006). Two more species, M. trionyxi Agrawal, 1966 from N. gangetica and M. lissemysia Chattervati, 1985 from L. punctata in India were synonymised with M. indica by Moravec et al. (2004). In addition, M. helicospicula Dey Sarkar, 2003 was described from Crocodylus palustris Lesson in India (Dey Sarkar, 2003), but its taxonomic position remains questionable (see below).

In this paper, we describe a new species of Micropleura from the Nile crocodile, Crocodylus niloticus Laurenti, in South Africa, using light and scanning electron microscopy (SEM).

Materials and methods

During July 2010 and February 2011, 16 Nile crocodiles were collected from various perennial rivers in the Kruger National Park, South Africa, as part of a study on their blood chemistry and disease ecology parameters. Nematodes found during post-mortem examination in the peritoneal cavity of four of these animals, were collected and stored in 70% ethanol. For morphological observation, specimens were cleared in lactophenol and studied as temporary mounts under a compound light microscope. First-stage larvae extracted from the uterus of a gravid female were studied in glycerine on slides, using the paraffin wax ring method. Specimens used for SEM were dehydrated through a graded ethanol series, immersed in hexamethyldisilazane for 10 min, air-dried, coated with gold in a JEOL JFS 1200 coater and examined using a JEOL JSM 5510 microscope at 10 kV. All measurements are in micrometres unless otherwise indicated. Metrical data are given as the range followed by the mean in parentheses. The oesophageal ratio, as defined by Moravec et al. (2004), is the length of the glandular part of the oesophagus vs the length of the muscular part. In the present specimens, the latter comprises the entirely muscular anterior portion and the posterior portion with a distinct dorsal glandular mass. The type-specimens have been deposited in the helminthological collections of the Museum für Naturkunde, Berlin, Germany (Entozoa ZMB) and the National Collection of Animal Helminths, ARC-Onderstepoort Veterinary Institute, South Africa (NCAH).


Family Micropleuridae Baylis & Daubney, 1922

Genus Micropleura von Linstow, 1906


Micropleura huchzermeyeri n. sp.


Type-host: Crocodylus niloticus Laurenti (Reptilia: Crocodylidae).

Type-locality: Type-specimens were collected from three crocodiles in the Kruger National Park, South Africa: One adult female from the River Crocodile (25°27′S, 31°58′E; 12.vii.2010; host no. 1), one subadult male from Silolveni Dam (24°49′S, 31°50′E; 15.vii.2010; host no. 2) and one adult female from the River Letaba (23°50′S, 31°38′E; 15.vii.2010; host no. 3).

Site in host: Peritoneal cavity.

Prevalence and intensity of infection: Overall prevalence of 25% (4 of 16 crocodiles examined); mean intensity 7.8 nematodes per infected host (range 1–25).

Type-specimens: Holotype: ZMB E.7615 (1 male; host no. 2). Paratypes: ZMB E.7616a-i (5 males and 4 females; host no. 2), ZMB E.7617a, b (2 females; host no. 1), NCAH.5.1 (6 males and 7 females; host no. 2), NCAH.5.2 (2 females; host no. 3), NCAH.5.3 (1 male stored in absolute ethanol; host no. 2).

Voucher-specimens: ZMB E.7619a (SEM stub, 1 anterior and 2 posterior fragments of males and 1 female; host no. 2), ZMB E.7619b (SEM stub, first-stage larvae extracted from the uterus of a single female; host no. 2), ZMB E.7618a-d and NCAH.5.4 (4 and 5 slides, respectively, containing first-stage larvae extracted from the uterus of a single female and mounted in glycerine, using paraffin wax ring method; host no. 2).

ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN, 2012), details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) for Micropleura huchzermeyeri n. sp. is urn:lsid:zoobank.org:act:1C8F5A35-94AF-4BC0-B086-DD4820CF520E.

Etymology: The species is named after the late Dr Fritz W. Huchzermeyer, University of Pretoria, in recognition of his invaluable contribution to our knowledge of the biology and diseases of crocodiles.

Description (Figs. 1–3)

General. Small-sized dracunculoid nematodes. Cuticle up to 3 thick, with very fine external transverse striations less than 1 µm apart; numerous elongate cuticular inflations, 5–30 long and c. 2–3 high, bearing minute papilla-like formations on their upper side, present on ventral and dorsal surface of body (Fig. 2E, F). Cuticular inflations starting approximately at level of midlength of muscular oesophagus, extending along entire length of body in females and terminating at anterior level of area rugosa in males. Inflations shorter, more boss-like towards anterior and posterior extremities, longer in mid-region of body. Inconspicuous lateral alae observed during SEM studies (Fig. 2A). Cephalic region rounded in lateral view, blunt in dorsoventral view (Figs. 1C, 2G). Oral opening minute, in the shape of a dorsoventrally elongated oval (Fig. 2C). Buccal capsule absent. Cephalic region bearing 14 papillae: 4 external sublateral papillae forming an outer, laterally elongated rectangle, 4 internal sublateral papillae forming an inner, laterally elongated rectangle and 4 submedian (2 subdorsal and 2 subventral) papillae forming a dorsoventrally elongated rectangle, as well as 2 lateral papillae positioned at level between external and internal sublateral papillae (Fig. 2C). Amphids situated just posterior to lateral papillae (Fig. 2B). Oesophagus distinctly divided into muscular and much longer, glandular part; muscular part further divided into cylindrical, anterior part, followed by posterior part with dilated mid-region containing well-distinct dorsal glandular mass (Figs. 1B, 2G). Nerve-ring surrounding oesophagus at base of muscular oesophagus (Fig. 1A, B). Deirids inconspicuous, club-shaped to conical, c. 4–5 long, situated posterior to nerve-ring and slightly anterior to excretory pore, at anterior level of glandular oesophagus (Figs. 1B, 2D). Tail with knob-like tip (Figs. 1F, I, 3C).

Fig. 1
figure 1

Micropleura huchzermeyeri n. sp. ex Crocodylus niloticus. A, Anterior extremity, female, left lateral view; B, Oesophageal region, female, right lateral view, note deirid (arrow); C, Head region, female, lateral (C1) and dorsoventral view (C2); D, Posterior extremity, male, right lateral view; E, Tail, male, right lateral view, note phasmid (arrow); F, Tail, male, ventral view, note phasmids (arrows); G, Gubernaculum, lateral view; H, Gubernaculum, ventral view; I, Posterior extremity, female, right lateral view; J, First-stage larva, lateral view

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Fig. 2
figure 2

Micropleura huchzermeyeri n. sp. ex Crocodylus niloticus. A, Anterior extremity, male, lateral view, note lateral ala and lateral papilla (arrow); B, Head region, male, lateral view, note amphid (white arrow), external sublateral papillae (white arrowheads), internal sublateral papillae (black arrowheads) and lateral papilla (black arrow); C, Head region, female, apical view, note external sublateral papillae (white arrowheads), internal sublateral papillae (black arrowheads), lateral papillae (black arrows) and dorsal and ventral submedian papillae (white arrows); D, Deirid, female; E, Detail of subventral cuticle with distinct inflations, male; F, Single cuticle inflation and fine transverse striations; G, Anterior extremity, male, lateral view (G1) and dorsal view (G2), note glandular mass in the posterior half of the muscular oesophagus (arrowhead)

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Fig. 3
figure 3

Micropleura huchzermeyeri n. sp. ex Crocodylus niloticus. A, Phasmid (arrow) and postanal subventral papilla (arrowhead) surrounded by fine cuticular tubercles, male, subventral view; B, Precloacal papilla (arrow) from first pair of papillae and detail of area rugosa composed of bands of fine cuticular tubercles (arrowheads), male, subventral view; C, Tail, female, ventral view, note phasmids (arrows) and cuticular inflations on the ventral surface; D, Phasmid, female, lateral view; E, Region of vulva (arrow) with atrophied vagina and split of two uteri (arrowhead), lateral view; F, First-stage larva, lateral view, note phasmid (arrow); G, Anterior extremity of first-stage larva armed with a larval tooth (arrowhead), lateral view; H, Phasmid (arrow), lateral view

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Male [Based on ten males.] Body 7.5–9.4 (8.4) mm long. Maximum width of body near midbody, 198–275 (236); body width at anus 109–154 (137). Total length of oesophagus, 1,358–1,837 (1,591), i.e. 17.9–20.6 (18.9)% of total body length; muscular oesophagus 303–404 (355) long, its anterior muscular part 173–225 (196) long, maximum width 57–77 (68), posterior part containing glandular mass 121–185 (158) long, maximum width 69–96 (87); glandular oesophagus 1,045–1,433 (1,236) long, maximum width 109–170 (137). Ratio of muscular to glandular oesophagus length 1:3.3–3.8 (3.5). Nerve-ring, deirids and excretory pore at 255–331 (295), 361–485 (437) and 386–570 (472), respectively, from anterior extremity. Reflexion of testis at about level of oesophago-intestinal junction. Tail curved ventrally, with translucent knob-like tip; 216–290 (254) long (Fig. 1D, F). Adcloacal alae 124–183 (157) long, restricted to short distance anterior and posterior to cloaca, with right ala slightly wider (Fig. 1E, F). Area rugosa 1,135–1,808 (1,362) long, extending anteriorly from approximately level of cloaca (Fig. 1D), comprising 30–41 (36) slightly elevated translucent bands with minute cuticular tubercles (Fig. 3B). Fine cuticular tubercles present around area of cloaca and first pair of subventral postcloacal papillae (Fig. 3A). Caudal papillae variable in size, composed of single ventral papilla at anterior rim of cloaca and 10 pairs of papillae arranged as follows: 2 pairs of large subventral precloacal papillae; 3 pairs of small subventral papillae surrounding cloaca; 1 pair of papillae just posterior to level of cloaca, right papilla on conspicuous lateral cuticular swelling, while papilla on left situated on ventrolateral surface without cuticular swelling; 4 pairs of papillae on tail, 2 subventral pairs at approximately midlength of tail, 1 lateral pair situated at level between the 2 subventral pairs, and a conspicuous pair of large lateral papillae on tail extremity (Fig. 1E, F). Phasmids lateral, immediately posterior to end of adcloacal alae (Figs. 1E, F, 3A). Spicules subequal, non-alate, very slender with sharply pointed tips; left spicule 577–673 (641) long; right spicule slightly longer, 614–722 (679) long. Gubernaculum well-sclerotised, 90–101 (97) long, with two prominent proximal lobes and rounded distal tip (Fig. 1G, H).


Female [Based on ten females containing first-stage larvae, except where otherwise indicated.] Body 18.2–41.3 (25.1) mm long. Maximum body width 407–1,131 (701); width near vulva 446–1,156 (653). Nerve-ring, deirids and excretory pore at 420–542 (477), 580–900 (698) and 640–960 (742), respectively, from anterior extremity. Total length of oesophagus 2,143–2,500 (2,334), or 6.1–12.3 (9.9)% of total body length; muscular oesophagus 499–648 (582) long, its anterior muscular part 287–398 (333) long, maximum width 119–175 (145); posterior part containing glandular mass, 212–274 (249) long, maximum width 134–210 (164); glandular oesophagus 1,644–1,852 (1,752) long, maximum width 153–279 (220). Ratio of muscular to glandular oesophagus length 1:2.7–3.3 (3.0). Rectum short, conical, with thick walls. Tail conical, straight to slightly curved ventrally, 227–362 (281) long (Fig. 1I). Phasmids on conspicuous protuberances at 139–234 (171) from tail tip (Figs. 1I, 3C, D). Vulva pre-equatorial, pore-like, situated at 8.0–17.2 (11.0; n = 9) mm from anterior extremity or 37.8–46.6 (43.4; n = 9)% of total body length; in one female 21.1 mm long, vulva at 12.1 mm from anterior extremity or 57.6% of total body length. Vagina directed posteriorly, its first part atrophied, 232–297 (n = 2) long, followed by part with lumen filled by hypotrophied epithelium, 222–421 (n = 2) long (Fig. 3E). Ovaries amphidelphic. In large females, uterus extending from level of midlength of muscular oesophagus to terminal part of intestine, filled with numerous first-stage larvae (Fig. 1A).


Immature female [Based on a single specimen.] Body 10.5 mm long. Maximum body width 332; width near vulva 321. Nerve-ring, deirids and excretory pore at 307, 470 and 588, respectively, from anterior extremity. Total length of oesophagus 1,616, or 15.5% of total body length; muscular oesophagus 379 long, its anterior muscular part 225 long, maximum width 83; posterior part containing glandular mass, 154 long, maximum width 109; glandular oesophagus 1,237 long, maximum width 148. Ratio of muscular to glandular oesophagus length 1:3.3. Rectum short, conical, with thick walls. Tail conical, straight to slightly curved ventrally, 163 long. Phasmids on conspicuous protuberances at 117 from tail tip. Vulva pre-equatorial, pore-like, situated at 4.7 mm from anterior extremity or 45.1% of total body length. Vagina directed posteriorly, its first part atrophied, followed by part with lumen filled by hypotrophied epithelium. Ovaries amphidelphic.


First-stage larva [Based on ten larvae extracted from the uterus of a gravid female.] Body 636–699 (663) long (Figs. 1J, 3F). Maximum body width 19–22 (20), width at anus 15–16 (15). Anterior extremity bearing larval tooth (Fig. 3G). Cuticle with distinct cuticular striation (Fig. 3H). Oesophagus 134–154 (144) long or 20.2–23.3 (21.7)% of body length. Nerve-ring at 73–114 (82) from anterior extremity. Tail 282–319 (294) long, or 42.3–45.6 (44.3)% of body length; filiform with pointed tip. Conspicuous phasmids situated just posterior to level of anus (Fig. 3H).

Discussion

The present specimens are assigned to Micropleura as defined by Moravec (2006), based on the presence of small elongate cuticular inflations on the body surface of both sexes, 14 cephalic papillae, a short muscular and long glandular oesophagus, a functional anus, a well-developed gubernaculum in males and the presence of an atrophied vulva followed by a much reduced vagina in the viviparous females. However, they differ from previously described Micropleura spp. in several morphological features. The main characters that distinguish M. huchzermeyeri n. sp. from all its congeners, are the length of its spicules, which clearly exceeds that of the remaining species and the tail with a knob-like tip in both sexes (Table 1).

Table 1 Metrical data of Micropleura spp. from various hosts and localities. Measurements in micrometres unless otherwise indicated
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In having 14 head papillae, ten pairs of caudal papillae and the presence of distinct cuticular inflations, a well-defined area rugosa in males and conspicuous phasmids on the female tail, the new species resembles M. australiensis, but can be distinguished from it by the presence of a conspicuous lateral cuticular swellings on the right side at the level of the cloaca bearing a papilla vs the absence thereof, the subequal spicules vs almost equal spicules in males and a tail with a knob-like tip in both sexes vs a tail with a rounded tip (Moravec et al., 2004, 2006). Two adcloacal alae are present in the current specimens, whereas an unpaired cuticular elevation resembling a caudal ala shifted towards the right side of the body has been described for M. australiensis (see Moravec et al., 2006). In addition, the vulva is postequatorial in M. australiensis, while it is typically pre-equatorial in M. huchzermeyeri n. sp. (but see single exception described above). The first-stage larvae of M. australiensis are distinctly shorter than those of the new species (Table 1).

The presence of three smaller pairs of papillae surrounding the cloaca is a character shared between M. huchzermeyeri n. sp. and M. vivipara as described by von Linstow (1906), but the latter species has shorter, equal spicules (Table 1), and large phasmids as seen in the females of M. huchzermeyeri n. sp. were not mentioned. Micropleura vivipara is described with a conspicuous lateral pair of papillae at the level of the cloaca, while in the new species only the right papilla situated just posterior to the cloaca is situated on a conspicuous lateral swelling. Moreover, von Linstow (1906) did not describe cuticular inflations as seen in the present specimens and recorded fewer tail papillae (four pairs and a single papilla vs ten pairs). First-stage larvae of M. huchzermeyeri n. sp. are longer than those of M. vivipara (Table 1). Subsequent to the description of M. vivipara by von Linstow (1906), a number of authors redescribed this species, either based on the type-material complemented by additional material (Baylis & Daubney, 1922) or on entirely newly collected material (Baylis, 1924; Maplestone, 1930). Both Baylis & Daubney (1922) and Maplestone (1930) described females assigned to M. vivipara as having conspicuous phasmids and cuticular inflations, features not mentioned by von Linstow (1906). Baylis (1924) assigned male specimens from a gharial to M. vivipara, but emphasised that they differed from males described by von Linstow (1906) in the absence of the prominent lateral papillae on the level of the cloaca, a conically tapering tail and the presence of a well-defined area rugosa. Having observed a papilla situated on a conspicuous swelling in the present material, it appears doubtful that the males examined by Baylis (1924) were indeed conspecific with those of von Linstow (1906). This conclusion is also supported by the differences in body length and spicule length between the specimens: 35.0 mm and 470 µm, respectively, in von Linstow’s (1906) specimens, vs 9.9–10.6 mm and 300–330 µm, respectively, in Baylis’s (1924) specimens.

Micropleura vazi, as described by Travassos (1933a, b), differs from M. huchzermeyeri n. sp. by the equatorial position of the vulva (vs pre-equatorial) and seven pairs of caudal papillae in males instead of ten observed in the present specimens. Furthermore, the first-stage larvae of M. vazi are distinctly shorter than those of M. huchzermeyeri n. sp. (Table 1). Moravec & Prouza (2003) supplemented the description of females of M. vazi based on specimens collected from C. crocodilus in Venezuela.

Micropleura indica differs from the new species in the absence of cuticular inflations, conspicuous phasmids on the female tail and large lateral caudal papillae in the male. In addition, the position of the vulva is equatorial in M. indica, not pre-equatorial as in M. huchzermeyeri n. sp. (see Khera, 1951; Siddiqi & Jairajpuri, 1963). First-stage larvae of M. indica are distinctly longer than those of the new species (Table 1).

A further species, M. helicospicula Dey Sarkar, 2003, was described from C. palustris Lesson in India (Dey Sarkar, 2003). However, the description, based on two males with a spicule length of 740–800 µm, is too perfunctory to ascertain their generic appurtenance. Consequently, we consider M. helicospicula a species incertae sedis. Nevertheless, the specimens differ from M. huchzermeyeri n. sp. in having longer spicules with spirally coiled proximal halves, a sharply pointed tail, seven pairs of caudal papillae in males and the absence of cuticular inflations.

Based on the above discrepancies between the morphology of the hitherto described species of Micropleura and the present specimens, as well as the geographical isolation of the latter in the Afrotropical region, we conclude that the specimens collected from C. niloticus in South Africa belong to a new species.

Species of Micropleura were not consistently described and currently different patterns of important taxonomical characters are recorded. Cuticular inflations have been described for four of the five species, M. vivipara sensu Baylis & Daubney, 1922, M. vazi, M. australiensis and M. huchzermeyeri n. sp. (see Baylis & Daubney, 1922; Travassos, 1933a, b; Moravec & Prouza, 2003; Moravec et al., 2004, 2006; this study). Cuticular inflations were also found in a single gravid female of Micropleura sp., reported by Moravec (2001) from Crocodylus moreletii Duméril & Bibron in Mexico and likely conspecific with M. vazi (see Moravec & Prouza, 2003). On the other hand, cuticular inflations appear absent in M. indica, the only species of Micropleura parasitising chelonian hosts (Khera, 1951; Siddiqi & Jairajpuri, 1963; Agrawal, 1966).

In the three species for which SEM studies of the apical view could be conducted, i.e. M. australiensis, M. vazi and M. huchzermeyeri n. sp., the number of cephalic papillae was confirmed to be 14, composed of four sublateral pairs, one lateral pair, one subdorsal and one subventral pair; furthermore, a pair of amphids is present posterior to the lateral papillae (Moravec & Prouza, 2003; Moravec et al., 2004, 2006; this study). An equivalent number of cephalic papillae was recorded and illustrated for M. indica (see Siddiqi & Jairajpuri, 1963). As these structures are difficult to study under a light microscope, it stands to reason that in M. vivipara, for which presently only six pairs of head papillae have been reported (von Linstow, 1906; Baylis & Daubney, 1922), not all papillae could be determined. Our study conforms with the assessment of Moravec et al. (2006) that the number of 14 head papillae is a generic characteristic of Micropleura.

The distinct subdivision of the muscular oesophagus, as observed in M. huchzermeyeri n. sp., had also been indicated in previously described species (Baylis & Daubney, 1922; Travassos, 1933a; Khera, 1951; Moravec & Prouza; 2003; Moravec et al., 2004), although the presence of a dorsal glandular mass in the posterior part had not been demonstrated. The glandular structure anterior to the nerve-ring of M. huchzermeyeri n. sp. might be homologous to the inflated glandular portion anterior to the nerve-ring which is considered part of the glandular oesophagus in members of the genus Dracunculus Reichard, 1759 (Dracunculidae Stiles, 1907) (see Moravec, 2006) and thus suggesting closer phylogenetic relationships between the two genera.

Large lateral caudal papillae supporting the caudal alae were found only in M. vivipara and M. huchzermeyeri n. sp. (see von Linstow, 1906; this study). Male spicules range from equal in M. vivipara and M. vivipara sensu Baylis, 1924 (von Linstow, 1906; Baylis, 1924), via almost equal in M. australiensis (Moravec et al., 2004) to subequal in M. indica, M. vazi and M. huchzermeyeri n. sp. (see Travassos, 1933a; Khera, 1951; Siddiqi & Jairajpuri, 1963; this paper).

Looking at Micropleura spp. females, the presence of large phasmids on the female tail has been reported in M. australiensis and M. huchzermeyeri n. sp. (see Moravec et al., 2004; this study), as well as in M. vivipara (see Baylis & Daubney, 1922). The position of the vulva varies from pre-equatorial in M. vivipara, M. indica and M. huchzermeyeri n. sp. (see von Linstow, 1906; Baylis & Daubney, 1922), via equatorial in M. vazi (see Travassos, 1933), to postequatorial in M. australiensis (see Moravec et al., 2004).

Species of Micropleura have been recorded from all three families of crocodilians, the Gavialidae, Crocodylidae and Alligatoridae. The finding of M. huchzermeyeri n. sp. in C. niloticus in South Africa is the first report of this genus in an Afrotropical host and constitutes an important expansion of its known geographical range. Thus, with the exception of the Nearctic, being home to Alligator mississippiensis (Daudin), representatives of Micropleura have now been reported from all zoogeographic regions that form part of the geographical range of its crocodilian hosts.