New Host and Locality Record of Parapharyngodon Japonicus (Nematoda: Oxyuroidea) from the Egyptian Changeable Lizard Agama Mutabilis (Agamidae): A Light and Scanning Electron Microscopy.

Parapharyngodon (Oxyurida) is a lizard gastrointestinal nematode parasite with a life cycle including lizards as main hosts. However, some species are known to parasitize anurans. In the present study, P. japonicus isolated from the large intestine of the Egyptian changeable lizard, Agama mutabilis was described and illustrated. Forty five specimens of these animals were collected from south Sinai desert, Egypt during the period from May to September 2017. After necropsy, the body was opened by a longitudinal incision from vent to throat, and the gastrointestinal tract was removed. The esophagus, stomach, small and large intestines were examined separately for helminthes. The recovered nematodes were examined by light and scanning electron microscopy. Thirty six specimens (80.0 %) were found to be naturally infected. The parasite was robust with prominent cuticular transverse annulations. Mouth surrounded by three bilobed lips, each with tiny labial papillae. Three pairs of caudal papillae were observed in male worms; 1 pair precloacal, 1 pair sublateral in cloacal opening line, 1 pair in proximal region of caudal appendage on its narrowed point. The posterior extremity beard dorsally directed caudal appendages. Females were with a conical posterior end terminated at a terminal spike. Ovaries reached esophageal isthmus but not wrapped around corpus. The parasite recorded was compared morphologically and morphometrically with the most similar species, it was found that it was most similar to P. japonicus with new host and locality records.

Introduction

The taxonomic status and validity of the genus Parapharyngodon have been questioned almost since its proposal by Chatterji (1933). Baylis (1936) considered it to be a synonym of the genus Thelandros Wedl (1861) (see Adamson, 1981; Adamson & Nasher, 1984; Bursey & Goldberg 1999, 2005). Later on, Freitas (1957) reinstated the genus Parapharyngodon which was accepted by several authors (Skrjabin et al., 1960; Baru & Coy-Otero, 1969; Baru, 1973; Sharpilo, 1976; Baker, 1987; Castazo-Fernandez et al., 1987; Ashour et al., 1994; Bursey & Goldberg, 2007a). Adamson (1981) re-established Parapharyngodon based on dietary habits of the host, morphology of the male genital cone and female tail and eggs. Males of Parapharyngodon spp. lack conical-shaped genital area and accessory piece. They have mammilliform papillae surrounding the more-or-less terminal anus and sub-terminal dorsally directed tail. Whereas males of Thelandros have a genital cone with pendulant papillae outside this cone, an accessory piece as well as terminal posteriorly directed tail is present in some species (Bursey et al., 2013; Pereira et al., 2017). Females of Parapharyngodon spp. possess a conical tail terminated in a short stout spike, eggs with sub-terminal operculum in the early stages of cleavage when released (Bursey et al., 2004; Anjum et al., 2013). Females of Thelandros have various tail morphologies, eggs with a terminal operculum larvated when released (Bursey et al., 2013; Pereira et al., 2017). More than 40 well described species are assigned to the genus Parapharyngodon according to Bursey and Goldberg (2007a, b) and Gupta et al. (2009). The Egyptian changeable lizard, Agama mutabilis Merrem (1820) is widespread across northern Africa, occurring from Western Sahara, Mauritania and Morocco east to Egypt and Sudan. This is an active predator exhibits a diurnal behavior and insectivorous including beetles, caterpillars and ants in diets and in some instances it has been known to eat large migratory locusts as they pass through its habitat. The present study reports the finding of P. japonicus in a new host, Agama mutabilis and locality, Egypt, including detailed morphological and morphometric characterization of this species using light and scanning electron microscopy (SEM).

Materials and Methods

Forty-five specimens of the mean snout-vents Agama mutabilis (length up to 9.4 cm) were collected by hand or noose from South Sinai desert, Egypt during the period from May to September 2017. Animals were subjected to euthanasia using 20 % benzocaine gel (Anbesol, Pfizer, Inc., New York). Each specimen was subsequently necropsied and all organs were examined for helminthes using a ZEISS Compact Greenough stereomicroscope (Model Stemi 305). All animal procedures were carried out according to the regulatory laws regarding experimental Animal Ethics Committee. Nematode worms were isolated from host intestines, heat fixed in 10 % neutral buffered formalin for 15 min and then preserved in 70 % ethanol in 5 % glycerol solution to avoid sudden drying. Finally, samples were transferred to lactophenol for clearance. The prepared samples were examined using differential interference contrast (DIC) light microscopy with digital image analysis system (analysis auto 5.0). Drawings were made with the aid of a camera lucida. Measurements were in micrometer unless otherwise stated. For SEM, samples were fixed in 4 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), washed in the same buffer, and dehydrated in a graded alcohol series. Samples were then processed in a critical point drier “Bomer–900” with freon 13, sputter–coated with gold–palladium in a Technics Hummer V, and finally examined with a Jeol scanning electron microscope (Model JSM7610F).

Ethical Approval and/or Informed Consent

All animal procedures were carried out according to the regulatory laws regarding experimental Animal Ethics and Use Committee.

Results

Thirty six out of 45 (80.0 %) specimens of the Egyptian changeable lizard, A. mutabilis were infected with nematode parasites isolated from their intestines. Worms were examined morphologically by light and SEM.

Parapharyngodon japonicus Houttuyn (1782)

Description based on 13 specimens (Figs. 1 – 3): Robust cylindrical nematodes with prominent cuticle annulations from beginning of the esophagus to the anal opening. Oral opening was triangular, surrounded by three bilobed lips. Each lobe beard tiny labial papillae. Buccal capsule absent. Sexual dimorphism evident, females larger and more robust than males. Lateral alae present in males, but absent in females. Males without caudal alae, caudal filament subterminal and directed dorsally. Females with conical tail terminated in a short stout spike.

Fig. 1

(A – E): Photomicrographs of P. japonicus showing: A – Females, lateral view with terminal spike (SK), TS transverse cuticle striations. B – Details of the cephalic end, mouth opening (M) surrounded by three bi-lobed lips. C – Transverse cuticle striations (TS). D – Posterior end of male with two spicules (SP). E – Posterior end of female terminated at a caudal spike (SK). Scale bars: A = 300 μm; B, C = 10 μm; D = 100 μm; E = 50 μm.

Fig. 3

Line drawings of P. japonicus. A, cephalic end of paratype female, apical view. B, Holotype male, lateral view. C, paratype female, lateral view. D, Posterior end of male, lateral view. L lips; OE oesophagus; IN intestine; TS transverse striations; TE testis; SV seminal vesicle; SP spicule; AO anterior ovary; PO posterior ovary; OD oviduct; AU anterior uterus; PU posterior uterus; A anus; PA caudal papillae. Scale bars: A = 10 μm; B = 200 μm; C = 300 μm; D = 100 μm.

(A, B): Scanning electron micrographs showing apical views of the cephalic end, L symbolized for the three bilobed lips (L1, L2, L3) surrounding mouth (M); TS for transverse cuticle striations. Scale bar = 10 μm.

Male: Small fusiform nematodes measured 1735 – 2986 (2280 ± 10) μm long, 385 – 490 (438 ± 11) μm wide at the level of the excretory pore. Lateral alae began at the level of esophageal isthmus. Total esophagus length 290 – 460 μm (388 ± 7). Bulb was 96 – 161 (140 ± 6) μm long and 65 – 105 (86 ± 5) μm wide. Nerve ring and excretory pore were 60 – 118 (70 ± 9) μm and 40 – 76 (46 ± 6) μm from the anterior end respectively. Testis reflexed posteriorly behind esophagus and the vas deferens separated from testis by a narrow tube. Three pairs of caudal papillae; 1 pair precloacal, 1 sublateral pair in cloacal opening line, 1 pair in proximal region of caudal appendage on its narrowed point. Spicules were 381 – 590 (550 ± 11) μm long. Posterior extremity of the body beard dorsally directed caudal appendages, terminated at a thin tip, 18 – 23 μm (20 ± 5) long.

Female: Body length 2150 – 3690 (2450 ± 17) μm long x 386 – 630 μm (510 ± 12) wide at the level of vulva. Esophagus length was 282 – 460 (375 ± 8) μm long; Bulb length 89 – 170 (153 ± 7) μm; bulb width 85 – 120 (96± 10) μm. Nerve ring and the excretory pore were at 64 – 110 (81 ± 10) μm and 42 – 86 (56 ± 7) μm from the anterior end respectively. Uteri were Amphidelphic and divergent; the anterior uterus directed anteriorly while the posterior uterus was posteriorly directed and joined at the mid body. Ovaries reached esophageal isthmus but not wrapped around corpus. Eggs were oval, slightly flattened, thin-shelled and with subterminal operculum. Asymmetrical eggs extracted from ovijector in the early stages of cleavage. The posterior end of female was conical with terminal stout spike 76 – 120 (90 ± 13) μm long.

Taxonomic Summary

Species: Parapharyngodon japonicus Houttuyn (1782)

Host: Agama mutabilis (Family: Agamidae)

Infection Site: Small intestine.

Locality: South Sinai, Egypt.

Prevalence: 36 out of 45 (80.0 %) specimens were infected

Deposition: Permanent slides of paratype female and holotype male were deposited at the Parasitology Division, Zoology Department Museum, Faculty of Science, Cairo University, Egypt.

Discussion

The nematode fauna of Egyptian reptiles has received little attention in recent years. The only studies include those of Seurat (1917), Baylis (1923) and Moravec et al. (1987); they described several species of nematodes from African reptiles, mostly of the genera Agama and Scincus (Table 1). The recorded species in the present study was assigned to the genus Parapharyngodon according to the key published by Bursey and Goldberg (1999) where species of Parapharyngodon are distinguished on the basis of the pattern of caudal papillae, morphology of the anterior cloacal lip, the location of the ovary, and geographical distribution. Worldwide, there are currently 83 nominal species have been assigned to Parapharyngodon, with sufficient morphological features for both male and female nematodes (Bursey & Goldberg, 2015; Ramallo et al., 2016). The parasite recorded herein can be differentiated from Thelandros sp. based on egg development during posture and the posterior end morphology in both sexes (Bursey & Goldberg, 2005). Parapharyngodon sp. males do not have a conical-shaped genital area, or an accessory piece. They have mammilliform papillae and a dorsal subterminal tail. Males of Thelandros sp. have a conical-shaped genital area with papillae disposed outside this cone (Bursey & Goldberg, 1999; Bursey et al., 2013). Females of Parapharyngodon sp. generally have a cone shaped tail with a thick pointed end, like a spike, eggs with a subterminal operculum that are un-cleaved, or in early stages of cleavage when released. In contrast, Thelandros sp. females have diverse tail morphology, eggs with terminal operculum, larvae are fully developed when they are released (Ramallo et al., 2016; Bursey et al., 2013; Velarde-Aguilar et al., 2015). Parapharyngodon japonicus recovered from Onychodactylus japonicus by Bursey and Goldberg (1999) in Japan is most similar to the present nematode isolated. Where both share the presence of postbulbar ovarian coils, the tail of female terminated at a small spike, eggs are thin-walled and the anterior cloacal lip is smooth. They resemble P. tyche in the presence of smooth anterior cloacal lip, ovary is postbulbar, and the eggs are thin-walled and oval in outline, the female spike is small and uterus is thick-walled. They differ from each other in that the spicules in P. japonicus male is half the length of those in P. tyche, and the lateral alae of P. japonicus end abruptly about 80 μm anterior to the cloaca, whereas in P. tyche, the lateral alae continue to the end of the body. All of the nematode species of family Pharyngodonidae reported from lizards in Egypt were compared (Table 1); two of them are from the same genus, P. bulbosus (Linstow, 1899) by Moravec et al. (1987) from Chalcides ocellatus in Egypt and P. micipsae. P. bulbosus differ from the present species in that tails of females which is conical without distinct caudal spike and ovarian coils not reaching anterior level of esophagus; while P. micipsae differ by the presence of postbulbar ovaries in females and their anterior ends forming prominent coils around the base of esophagus while their males have 4 pairs of caudal papillae and echinate anterior cloacal lip. Moravec et al. (1987) isolated a nematode P. aegyptiacus which further has since been transferred to Skrjabinodon inglis by Moravec and Barus (1990).

Table 1

Comparative measurements between the present species and members of family Pharyngodonidae previously reported from lizards in Egypt, measurements in mm, otherwise stated.

Species	Host	locality	length	width	Spicule (mm)	Cloacal lip	Ovary	Egg size	Reference
T. alatus (Wedle, 1862)	Agama stellio	El Quseima, North Sinai	5.94	0.44	-	-	-	14X(72-84) μm	Edward A. Belle, 1954
T. kasauli (Chatterji, 1936)	Agama stellio	El Quseima, North Sinai	Female: 5.23 Male: 3	Female: 0.35 Male:0.31	100 μm–	-	-	128X68 μm	Edward A. Belle, 1954
T. bulbosusv. (Linstow, 1899)	Agama- Scincus	Berg el Arab (Agama), Wadi Faran, S.Sinai (Scincus)	Female 3.8- 4.61 Male: 2.6	-	74-79 μm	-	|Prebulbar	97X57 μm	EdwardA. Belle, 1954
T. micipsae (Seurat, 1917)	Chalcides sepoides	Zawiet,Abu Musa1 lam, Giza	Female: 6.90-7.0 Male: 2.30	Female: 8.0 Male: 0.17	74 μm	-	Postbulbar	50X90 mm	Edward A. Belle, 1954
T. cameroni Edward A. Belle 1954	Chaleides sepoides - Scincus	Berg el Arab, W. Desert, Kom. Aushdm, Faiyam Province	Female:3.28 Male: 2.34	Female: 0.31 Male:0.20	72 μm	-	Postbulbar	79-95 X 52-54 μm	Edward A. Belle 1954
T. kuntz (Edward A. Belle 1954)	Agama	Wadi Faran, S. Sinai	Female:3.20-4.20 Male:2.23-2.65	Female:0.39 Male:0.20	50 μm–	-	-	100 X 58 μm	Edward A. Belle, 1954
Thelandros sp.	Chalcides ocellatus Chalcides sepsoides	S.Sinai	Female: 2.65 -3.85 Male:1.85 - 3.02	Female: 0.36 -0.46 Male:0.17- 0.25	-	-	Prebulbar	78 -84x51 -68 μm	Rabie et al, 2012
Pharyngodon hindlei (Thapar, 1925)	Eumeces schneiderii	Berg El Arab, W. Desert.	Female:4-5 Male:2.5-0.3	Female:0.4-0.47 Male:0.16	0.045-0.054	-	-	140 X 42-51	Edward A. Belle, 1954
Pharyngodon extenuates (Rudolphi, 1819)	Acanthodactylus	Baltim, Fouadiya Province,	Female:5-6.7	Female:0.36	-	-		144 X 33-36	Edward A. Belle, 1954
Pharyngodon inermicauda (Baylis, 1923)	Tarentola mauritanica	Abu Rawash	Femal:3.60-3.81 Male:1.46- 1.84	Female:0.326-0.340 Male:0.095-0.163	absent	-	In the middle third	0.150-0.165 X 0.042-0.51	Moravec et al.,1987
Pharyngodon mamillatus (Linstow, 1897)	Chalcides ocellatus	Abu Rawash	Female:3.26-3.63 Male:1.07-2.23	Female: 0.340-0.394 Male:0.109-0.204	0.033-0.045	-	In the middle third	0.135-0.144 X 0.036-0.042	Moravec et al., 1987
Ρ bulbosus (Linstow, 1899)	Chalcides ocellatus	Abu Rawash	Female:2.86-4.28 Male:2.14-2.46	Female:0.340-0.449 Male:0.231-0.236	0.051-0.063	smooth	Postbulbar	90-99 X 54-57	Moravec et al., 1987
Ρ micipsae (Seurat, 1917)	Scincus scincus	Abu Rawash	Female:4.46-0.6.77 Male:1.71	Female:0.503-0.830 Male:0.095mm	88 μm	echinate	prebulbar	91 X 50	Moravec et al., 1987
Ρ Japonicus (Present study)	Agama mutabilis	S. sinai	Female: 2150-3690 μm Male:1735-2986 μm	Female: 386-630 μm Male:385-490 μm	381 -590 μm	smooth	Postbulbar	76- 120μm