Intestinal Helminth Parasites of Caspian Turtle Mauremys Caspica (Gmelin, 1774) (Testudines, Geoemydidae) from Al-Diwaniya Province, Iraq

Shubber et al (2020): Prevalence of intestinal helminth parasites of Caspian turtle August 2020 Vol. 23 Issue 12

Intestinal Helminth Parasites of Caspian Turtle Mauremys Caspica (Gmelin, 1774) (Testudines, Geoemydidae) From Al-Diwaniya Province, Iraq

Habeeb W. Kadhum Shubber * Ali B. Mohsen Alwaaly* and Mohammad K. Mohammad**

*Department of Biology, College of Science, University of Al Qadisiyah **Department of Medical Lab Science, College of Health and Medical Technology, Uruk Private University, Baghdad, Iraq *Corresponding author: [email protected]

Abstract: The caspian turtle Mauremys caspica (Gmelin, 1774) is a medium-sized semi aquatic freshwater turtle. It is widely distributed in suitable areas throughout Iraq. A total of 28 road-killed specimens have been examined for their intestinal helminthic parasites. Results have been showed that 78.6% of this turtle were infected with one or more of following parasite species of Telorchis assula (Trematoda), Falcaustra araxiana, Serpinema microcephalus, and Contracaecum sp. (Nematoda).The statistical analysis showed that there were significant differences in prevalence between male and female turtles in parasitic infections (P<0.05) as the percentage of females was higher than males. Morphometric and meristic description for the helminthes have been provided. Reporting the nematode F. araxiana from M. caspica in this study constitutes a new record for Iraqi parasitic fauna as well as a new host record. It also expands the geographical distribution of this parasite to include Armenia, Iran and Iraq. A description based on measurements and number of these worms was given.

Keywords: Al-Diwaniya, Caspian turtle, Contracaecum sp., Falcaustra araxiana, Iraq

How to cite this article: Shubber HWK, Alwaaly ABM, Mohamed MK (2020): Intestinal helminth parasites of Caspian turtle Mauremys caspica (Gmelin, 1774) (Testudines, Geomydidae) from Al-Dawaniya Province, Iraq, Ann Trop Med & Public Health; 23(S12): SP231237. DOI: http://doi.org/10.36295/ASRO.2020.231237

1. Introduction The caspian turtle Mauremys caspica (Gmelin, 1774) was recorded from Iraq by Khalaf (1959) as Clemmys caspica caspica. Al-Barazengy et al. [1] reported two subspecies for Iraq M. c. caspica and M. c. siebenrocki without mentioning the local distribution. It is a medium-sized freshwater turtle in the eastern Mediterranean region [2,3] ; it is widely distributed in suitable areas especially in permanent water bodies throughout Iraq in considerable numbers in central and southern parts. This turtle tolerates moderate salinity level of water since it presents in large numbers in marshy areas with brackish water such as Al-Huwaiza marsh (south) and Al-Dalmaj Marsh (central) [4].Only few works carried out in Iraq in regard to parasites of this turtle; [4] examined 30 specimens collected in Erbil, Sulaimaniya and Kirkuk provinces in the north of Iraq. They found three species of nematodes, Atractis dactyluris, Camallanusmicrocephalus and Tachygone trianicollei. [5] isolated Telorchis cyclemmidis from this turtle in Al-Ramadi city, Al-Anbar Province, Western Iraq. [6] examined 30 specimens of this turtle in the north of Iraq and recorded both the nematode Camallanus microcephalus and the trematode Telorchis stunkardi. [7] reported Chabaudinema americana from turtles collected in Basrah city in the south.The aim of the present work is to detect identification, prevalence, and intensity of the intestinal parasitic helminthes in the caspian turtle collected in Al-Diwaniya Province, central Iraq.

2. Materials and Methods Study area: Al-Diwaniya Province is located in the middle-Euphrates region (Map 1). It is known to be a very well irrigated and is considered to be one of the most fertile parts of Iraq which maintains a wide range of ecological areas, including wetlands, arid zones, agricultural areas, as well as semi-desert regions. The average high temperature is 30°C and the average low temperature is 17°C with a total area of 8,153 km2 represents about 1.9% of Iraq [8]. Sampling: A total of 28 (11 males and 17 females) road-killed specimens were collected from the period April to September 2015 in suitable aquatic areas in Al-Diwaniya Province, middle of Iraq including the marshy areas of Al- Dalmaj and Al-Sanniya areas. The turtles were transferred to the lab in the College of Science, Al-Qadisiya University in Al-Diwaniya city as soon as possible, dissected and their intestines were carefully searched for helminth parasites. The recovered helminthes were treated following [9]. Trematodes were cleaned by washing thoroughly with normal saline, killed and kept in 70% alcohol. Some trematodes were stained with acetocarmine and passed through a series of concentrations of 70%, 80%, 90% and 100% alcohol for dehydration, cleared in xylene and mounted in Canada balsam. The nematodes were first put in worm saline and then transferred to a mixture of 70% alcohol (95%) and glycerol (5%) for fixation and preservation. On examination, they were cleared with lactophenol. Photomicrographs were taken with

Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.231237

Shubber et al (2020): Prevalence of intestinal helminth parasites of Caspian turtle August 2020 Vol. 23 Issue 12

the aid of HDCE-X5 digital camera. Scale bar equal to 1 mm (exception plate 9, the scale bar equal 0.25 mm.). Measurements in millimeter (mm) and presented as mean followed by standard deviation and range between parentheses.

Map (1): The map of Iraq showing situation of Al-Diwaniya Province, Central Iraq. (Iraq-Administrative Maps, GlobalSecurity.org). 2.1 Statistical analysis: The results from male and female turtles were analyzed with Chi Square test at (P<0.05) level.

3. Results and Discussion

The inspection of caspian turtle Mauremys caspica revealed their infection with four species of parasites. The number and sex of parasitic helminthes recovered from the infected Mauremys caspica specimens are summarized in Table (1).The results revealed that 22 (78.6%) of M. caspica were infected with either Telorchis assula (Trematoda), Falcaustra araxiana, Serpinema microcephalus or Contracaecum sp. (Nematodes). Females acquired general infection rate of 82.4% while males acquired general infection rate of 72.7% and showed significant differences in prevalence between male and female turtles (P<0.05). The percentage of infection for females was higher than those of males (X2=13.876). This result agreed with the findings of Youssefi et al. [10] who found a significant variation in the infection rate between the two mates emphasizing that females had higher parasitic prevalence than males. However, the percentages of infection in the two studies are quite different reflecting the environmental potentiality differences between the two studied areas. In Brazil, Pereira et al. [11] found the same case with the freshwater turtle Phrynops geoffroanus (Chelidae) and attributed their results to the differences in sexual dimorphism related to body stating that this must have contributed to a higher parasite load in female hosts; pattern of infection in females reveals that the single mode were 63.6%, double 27.3% and triple 9.1%, while in males the single and double mode was 50% each.

Table (1): Number and sex of parasitic helminthes recovered from the infected Mauremys caspica specimens.

Hosts (n=28) Mean of Number Helminthes Number of Number of Total percent intensity of infected infected (range) infected Telorchis assula 8 3 (27.27) 5 (29.41) 28.57 7.75 (1-22) Falcaustra araxiana 16 7 (63.63) 9 (52.94) 57.14 15.81 (1-55) Serpinema 10 2 (18.18) 8 (47.06) 35.71 4.3 (1-6) microcephalus Contracaecum sp. 1 X 1 (5.88) 3.57 1 (1) Total * 22 72.72 82.35 78.57

X2=13.876 *This number represents the number of hosts that have single infection regardless the specie of parasite

The following is an account on the description and measurements of these parasites: Telorchis assula (Dujardin, 1845) (Digenea, Telorchiidae) (Pls. 1-3). This helminth infects 28.57% of the total sample of hosts with an intensity and range 7.75 (1-22) (Tab. 1).

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Description: Body elongate, 9.96±2.3 (7.74-13.62) long by 0.685±0.147 (0.56-0.98) width at acetabulum level; tegument has minute spines along the whole body. This helminthes has oral sucker subterminal, circular to subcircular, without lappets, 0.164±0.013 (0.154-0.180) long by 0.146±0.018 (0.116-0.180) width. Pharynx is muscular, compact and short 0.133±0.028 (0.075-0.150) X 0.112±0.018 (0.072-0.128), esophagus 0.268±0.043 (0.178-0.312) long, caeca extending to 0.115±0.028 (0.050-0.163) near posterior end of the body. The acetabulum is weakly developed, almost circular,0.133±0.011 (0.118-0.163).Testes are two, oval, tandem, contiguous, intercaecal, anterior testis 0.383±0.103 (0.252-0.528) long by 0.293±0.078 (0.152-0.404) width, posterior testis 0.472±0.153 (0.276-0.652) long by 0.298±0.077 (0.176-0.404) width; cirrus sac is 2.868±0.272 (2.604-3.478) in length with coiled seminal vesicle; genital pore situated between acetabulum and bifurcation; ovary situated slightly behind the middle of the body, almost rounded 0.258±0.053 (0.206-0.354) in diameter; descending and ascending loops of uterus confined to intercaecal space; vitellaria lateral, extracaecal, between anterior testis and midway between anterior extremity of uterus and acetabulum; eggs are oval 0.0293±0.0023 (0.0250-0.0325) long by 0.0143±0.0022 (0.0100-0.0175) width. Excretory vesicle Y-shaped, arms short, excretory pore terminal.

Plate (1): Anterior part of the trematode Telorchis assula.

Plate (2): Middle part of the trematode T. assula.

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Shubber et al (2020): Prevalence of intestinal helminth parasites of Caspian turtle August 2020 Vol. 23 Issue 12

Plate (3): Posterior part of the trematode T. assula.

Members of the genus Telorchis are known to inhabit the intestines of salamanders, turtles, and snakes worldwide [12]. Natrix natrix and N. tessellata find in Baghdad City [13] ; furthermore, some reports that T. cyclemmidis and T. stunkardi from M. caspica caspica in west and north of Iraq respectively [14,15]. In Iran, it infected N. natrix, N. tessellata, and Mauremys caspica caspica in the north of Iran [16-19], while it is reported from Emys orbicularis in the southwest [20]. Some reportstalk this parasite from N. natrix in Iran without mentioning its collection site [21]. Yildirimhan et al. [22] found it in N. natrix and N. tessellate in Turkey. It was reported also from N. natrix in Mordovia [23] , and from N. tessellate in Armenia [24]. In the present study, the infection prevalence was moderate and reached 28.6% compared to high infection rate in the snakes. In the present study although the infection rate was moderate and reached 28.6% but it still far from that reported in snakes. This may suggests more successful parasitism in snakes rather than in turtles. Relatively lower infection rate with T. assula was recorded in Mauremys caspica compared to high infection rate in the snakes Natrix natrix and N. tessellate; for example, Yildirimhan et al. [22] in Turkey found infection rate of 52% and 71% in N. natrix and N. tessellata respectively. In Iran Halajian et al. [24] recorded 100% infection rate with T. assula in Natrix natrix while Yousesfi et al. [25] found that infection in M. caspica was only 2.9% compared to 84.6% and 50% in N. natrix and N. tessellate respectively; in Iraq Al-Moussawi [2] found infection rates 62.5% and 80% in N. natrix and N. tessellata respectively. On the other hand, mean intensity of T. assula varied widely among hosts and collection sites; Yildirimhan et al. [22] founded mean intensity of 4.63 and 7.35 for N. natrix and N. tessellata respectively. Halajian et al. [24] recorded intensity of 101.7 for N. natrix. Al-Moussawi [2] reported 1.54 as collective mean intensity in both N. natrix and N. tessellata. Results of this study showed that intensity was 7.75. This variation comes as a result of examining different hosts, countries, ecosystems, and intermediate hosts' potentiality as well as the difference in sample size of hosts examined among the previous studies. Falcaustra araxiana Massino, 1924 (Nematoda, Kathlaniidae)The general prevalence of males and females of Mauremys caspica with F. araxiana was 57.14%. Infection rate for males was 63.63% and 67.86%; the following is an account on the description and measurements (based on ten specimens for each species) of these parasites which were recorded here for the first time in Iraq. Description: (10 specimens) (Pls. 4-7); the body is cylindrical tapers posteriorly and truncates anteriorly; mouth area is nearly circular, diameter 0.15, with three lips, buccal cavity rectangular 0.07x0.13, distance from anterior extremity to the nerve ring 0.13, pharynx is well defined and transversally striated with total length of 2.65±0.504 (2.6-2.83) composed of three parts, anterior glandular with length of 2.36±0.8806 (2.28-2.48), muscular oblong isthmus with length of 0.20 and width of 0.15 directly anterior to the sub-spherical esophageal bulb with length of 0.3 (Pl. 4).

Male: Total length 13.8±2.008 (13.3-14.3), maximum width 0.48±0.022 (0.45-0.50). Anus situated at 0.57 from posterior extremity; spicules are strongly curved, prominent, thick and equal in size with length of 2.83 ± 0.771 (2.75- 2.88) (Pl. 5), pseudosuckers 4 (Pl. 6), rather ellipsoid, resemble each other, the first pseudosucker (nearer to anterior end) 0.388±0.102 (range 0.375–0.4) in long and 0.156±0.045 (range 0.125–0.175) depth; the second: 0.406±0.114 (0.375-0.45) and 0.169±0.058 (0.15-0.175); the third: 0.381± 0.161 (0.325-0.425) and 0.169 ± 0.066 (0.15-0.2); the fourth: 0.413 ± 0.102 (0.375-0.45) and0.172 ± 0.118 (0.15-0.2). Each pseudosucker is supported by approximately 24- 30 radially arranged muscle bands. Posterior lip of the fourth pseudosucker is 3.092±1.122 (2.95-3.2) from tip of tail; papillae pattern is 6-6-10+1. Ten pairs of caudal papillae situated posterior to pseudosuckers.

Female: total length 16.13±2.556 (16-16.30), maximum width 1.41±0.116 (1.38-1.44), vulva in the posterior half situated at 6.06±1.063 (5.88-6.25) from the posterior extremity, anus situated at 1.50 ± 0.173 (1.38-1.63) from posterior extremity (Pl. 7), eggs are ovoid 0.013±0.007 (0.010-0.016) x 0.008±0.0006 (0.006-0.009).

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Plate (4): Anterior end of Falcaustra araxiana.

Plate (5): Posterior end of male F. araxiana.

Plate (6): The four pseudosuckers (arrows) on the ventral surface of male specimen of F. araxiana.

Plate (7): Posterior end of female F. araxiana. The species of the genus Falcaustra are parasites of fish, amphibians and reptiles [26;27] with a total of 95 nominal species reported worldwide [28]. F. araxiana has typical esophagus of Family Kathlaniidae and Subfamily Kathlaniinae. Nevertheless, variations in pharynx and in male caudal structures cause difficulties in classifying this family, for example genus Falcaustra usually has a typical pharynx with spherical isthmus which may become cylindrical, or subspherical as the case in the present specimens, in certain species where there have been fixed in an extended position [29]. In the present study, two forms of F. araxiana were noticed, the pharynx in the first form is simple with distinct isthmus and bulb while in the second form is rather coiled especially at posterior area and the isthmus and bulb are not differentiated. Males of the first form with relatively longer spicules, four weakly defined pseudosuckers, the posterior end weakly curved and eggs in females of the same form are slightly smaller; males of the second form have relatively shorter spicules, four well defined pseudosuckers, and the posterior end very strongly curved, in females of the same form eggs are rather larger. After a correspondence with Prof. Charles Bursey, Pennsylvania State University, Pennsylvania, USA on 30.6.2015, these two forms considered as variations within a species, the slight differences in spicules and pseudosuckers may reflect age differences or perhaps even physical damage to the worm during grown in the turtle’s intestine. The pharynx differences may be a fixation artifact. This species could be differentiated from the closely related F. armenica in attaining total length and spicule length more than twice than its counterpart and in having four pseudosuckers instead of one [30,31]. Yamaguti stated that the European pond turtle is the only known host for this nematode species. Shayegh et al. [31] found that it is the most prevalent nematode in European pond turtle Emys orbicularis in southwest Iran and noticed that there is no information about life cycle and geographical distribution of this species. The reporting of F. araxiana from M. caspica in this study

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Shubber et al (2020): Prevalence of intestinal helminth parasites of Caspian turtle August 2020 Vol. 23 Issue 12

constitutes a new record for Iraqi parasitic fauna as well as a new host record; it also expands the geographical distribution of the parasite to include Armenia, Iran and Iraq. However, Youssefi et al. reported F. armenica from M. caspica in northern Iran.In Iraq, examined 30 and 45 specimens of M. caspica respectively collected in northern parts of Iraq but surprisingly both studies could not record F. araxiana; the mean intensity of this parasite in the present study is 15.81 [32,33] (Tab. 1); Shayegh et al. [31] found an intensity of 18 in Emys orbiularis in Iran; this difference may be a result of the presence of the parasite in rather two widely different taxa.

Serpinema microcephalus (Dujardin, 1845) (Nematoda, Camallanidae)

The general prevalence of males and females of Mauremys caspica with S. microcephalus was 35.71%. Prevalence for males was 18.18% and 47.06% in females. Present result on prevalence is almost similar to that recorded by Molan and Saeed [2] but with lower intensity. They examined 30 specimens of Mauremys caspica collected in Hareer (Erbil), Sarjinar (Sulaimaniya), Alton-Kopri (Kirkuk) and Rabia'a (Mosul) with an overall infection rate of 30% and mean intensity of 4 worms / host.

Description: the living worm is red in color with smooth cuticle; mouth opening is slit-like. Anterior muscular pharynx clearly differentiated from posterior glandular part (Pl. 8). Cephalic extremity (Pl. 9) with laterally compressed brown buccal capsule composed of 2 valves and a basal ring. Valves are with longitudinal ridges. Anterior margin of valve with 2 elongated plates. Buccal valves are supported by dorsoventral tridents on each side.

Males (5 specimens): Body length 12.020 ± 0.559 (11.295-12.650) and maximum body width 0.319 ± 0.041 (0.282- 0.370). Buccal capsule includes basal ring 0.147 ± 0.028 (0.128-0.170). length of tridents is 0.070 ± (0.050-0.102); muscular esophagus is 0.464±0.016 (0.46-0.47) long and 0.107 ± 0.033 (0.090-0.120) maximum width. Glandular esophagus is 0.848 ± 0.115 (0.720-0.970) long and 82–139 wide. The posterior end of body (Pls. 10-11) has relatively broad caudal alae supported by pedunculate papillae and opaque alae, tail 0.383 ± 0.069 (0.308-0.460). Caudal papillae: 10 pairs of preanal and 6 pairs of postanal slender, pedunculate papillae present. Spicules are unequal; the large one is 1.119 ± 0.102 (1.088-1.155) while the small one 0.752 ± 0.048 (0.682-0.800); tail conical with rounded tip, 0.383±0.069 (0.308-0.460) long.

Females (5 larvigerous specimens): Body length 23.218 ± 4.658 (16.429-26.183) long, maximum body widths 0.485±0.085 (0.385-0.518). Buccal capsule is 0.141 ± 0.015 (0.128-0.154); muscular pharynx is 0.670 ± 0.192 (0.470- 0.924), glandular pharynx 0.768 ± 0.158 (0.550-0.924). In most specimens, vulva (Pl. 12) postequatorial situated a little after middle of the body but in few specimens it was slightly preequatorial or equatorial, 11.546 ± 1.952 (9.113-13.862) from anterior end of body; vagina directed anteriorly. Uterus filled with larvae (Pl. 13). Tail conical is 0.432 ± 0.024 (0.411-0.460) long (Pl. 14).

Plate (8): Anterior end of Serpinema microcephalus.

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Plate (9): Cephalic extremity showing ridges in buccal valves of S. microcephalus.

Plate (10): Posterior end of male S. microcephalus with protruded spicules.

Plate (11): Posterior end of male S. microcephalus.

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Shubber et al (2020): Prevalence of intestinal helminth parasites of Caspian turtle August 2020 Vol. 23 Issue 12

Plate (12): Vulva region of female S. microcephalus.

Plate (13): Trunk region of viviparous female S. microcephlus filled with newly hatched nematode larvae within the mother body.

Plate (14): Posterior end of female S. microcephalus.

Baker [34] stated that S. microcephalus is a parasite of turtles in western Palaearctic realm. It is proved later that this parasite has wider geographical distribution as well as diverse range of hosts. S. microcephalus was recorded in Iraq [35], in Spain [36]; in Turkey [37]; in Iran [31]. Species of this genus as the present nematode has a unique cephalic structure with buccal valves and viviparous reproduction pattern (Pls. 10 and 13). It was reported for Iraq as Camallanus microcephalum from the Caspian turtle by [38] widely geographical distributed in the north of Iraq but with relatively low prevalence. This nematode was frequently recorded in various freshwater turtle species, in Emys blandingii [39]; Chrysemys picta, Chelydra serpentina, Clemmys insculpta and Emys blandingii [40] ; Emys orbicularis [41]; Chrysemys picta [42] ; Mauremys leprosa [43] ; Trachemys scriptaelegans [37] ; Trachemys scripta and Pseudemys concinna [27] and Mauremys caspica [28] . The mean intensity of this parasite in the present study is 4.3 (Tab. 1), while it was 3 in the study of Shayegh et al. [31].

Contracaecum sp. larva(Pls.15, 16): A single specimen of larval stage of Contracaecum sp. was found in the intestine of one of the female hosts examined.

Description: Total length 24.2 and the maximum width 1.18. Buccal cavity is 0.1 x 0.19; well transversally striated on whole body; there is an appendix on the end of the esophagus. Contracaecum larvae could not be identified to species level because the reproductive organs of the worms, which are used for characterization, were not yet fully developed [43].Contracaecum larvae are often found in abnormal hosts and do not develop to adults in turtles. Moreover, Contracaecum larvae show low specificity. In view of these facts it could be concluded that this turtle host may eat fishes in their aquatic ecosystems. Turtles older find than one-year feed on a wide range of animals. These food habits provide a chance for incident transfer of the parasite to the turtle host [5].

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Plate (15): Anterior end of Contracaecum sp. larva.

Plate (16): Posterior end of Contracaecum sp. larva.

Acknowledgements

The authors would like to express their deep gratitude to Dr. John Mike Kinsella, USA for identifying the trematode specific identity. We are also grateful to Mrs. Khalida I. Hasson and Mr. Osama M. Kadhim from the Iraq Natural History Research Center and Museum, University of Baghdad for their help in lab work and manuscript preparation.

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