Turkish Journal of Zoology Turk J Zool (2018) 42: 152-157 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Short Communication doi:10.3906/zoo-1705-61

Natural infection of the land snails obvia, detrita, and Monacha samsunensis by Brachylaima sp. metacercariae in Kastamonu, Turkey

Fatma Mohamed Abdualrazig Khalifa ISMAIL, Gözde GÜRELLİ* Department of Biology, Faculty of Sciences and Arts, Kastamonu University, Kuzeykent, Kastamonu, Turkey

Received: 29.05.2017 Accepted/Published Online: 23.10.2017 Final Version: 10.01.2018

Abstract: This study investigated the prevalence of infection, and some of its morphological features, of the land snails , , and Monacha samsunensis (: ) by metacercariae of Brachylaima sp. (: ) in Kastamonu, Turkey. The land snails were collected in spring and autumn. The prevalence rates of infection by metacercariae of Brachylaima sp. were 5.74%, 1.68%, and 17.35% in X. obvia, Z. detrita, and M. samsunensis, respectively. This study reports for the first time the natural occurrence of Brachylaima sp. unencysted metacercariae in the hepatopancreas of these three land snails.

Key words: Brachylaima sp., metacercariae, Monacha samsunensis, Xerolenta obvia, Zebrina detrita, Turkey

Brachylaimidae Joyeux & Foley, 1930 is a large family of The goal of this study was to investigate the prevalence digenean trematodes comprising several genera and and morphological features of infection of the land with a worldwide distribution. The Brachylaima snails Xerolenta obvia, Zebrina detrita, and Monacha Dujardin, 1843 is a member of this family and comprises samsunensis (Mollusca: Pulmonata) by the metacercariae the primary endoparasites of endothermic vertebrates, of Brachylaima sp. (Trematoda: Brachylaimidae) in including birds, mammals, and humans (Yamaguti, 1975; Kastamonu, Turkey. Guisantes et al., 1994; Butcher and Grove, 2001, 2005; The land snails X. obvia, Z. detrita, and M. samsunensis Lunaschi and Drago, 2012; Reda and El-Shabasy, 2016; were picked up following rainfall from areas in Kastamonu Suleman and Khan, 2016). Land snails and slugs are Province in April and May (spring) and October and required as the first and second intermediate hosts in the November (autumn) in 2016. A total of 383 specimens of life cycle of Brachylaima spp. (Yamaguti, 1975; Butcher and X. obvia, 119 specimens of Z. detrita, and 121 specimens Grove, 2005; Barger and Hnida, 2008; Barger, 2011; Sirgel of M. samsunensis were picked up. In the laboratory, et al., 2012; Valente et al., 2016). First intermediate hosts the land snails were dissected while alive and their have sporocysts and cercariae, and second intermediate hepatopancreases were taken out. These organs were hosts have metacercariae of Brachylaima spp. Infection spread on clean glass slides with 0.6% NaCl solution. occurs when the definitive hosts consume raw mollusks Using a mounted needle, the hepatopancreas was incised containing metacercariae (Butcher and Grove, 2001, and a thin film of the liquid was dropped onto a slide for 2005; González-Moreno and Gracenea, 2006; Barger and investigation of parasites under a light microscope. After Hnida, 2008; Barger, 2011; Sirgel et al., 2012; Gracenea and the metacercariae of Brachylaima sp. were identified, they Gállego, 2017; Nakao et al., 2017). were fixed in 5% formalin. The fixed parasites were coated Although some studies have been conducted on the with an adhesive mixture and stained with Semichon’s second intermediate host mollusk species of Brachylaima acetocarmine. spp. worldwide (Thiengo and Amato, 1995; Barger and Some samples were prepared for scanning electron Hnida, 2008; Barger, 2011; Sirgel et al., 2012; Valente microscope (SEM) examinations, for which the formalin- et al., 2016; Gracenea and Gállego, 2017; Nakao et al., fixed specimens were washed in distilled water overnight 2017), only sporocysts and cercariae have been reported and postfixed in 2% (w/v) osmium tetroxide aqueous in Helix aspersa as a first intermediate host in Turkey solution for 4 h at room temperature. After 5 washes with (Köse et al., 2015); metacercariae have not been reported. distilled water, the specimens were dehydrated through

* Correspondence: [email protected] 152 ISMAIL and GÜRELLİ / Turk J Zool an ethanol series, kept for 20 min in each concentration, of the total 119 Z. detrita specimens, and 21 specimens and dried in a CO2 critical-point dryer (Imai et al., 1992). (17.35%) of the total 121 M. samsunensis specimens (Table The dried specimens were sputter-coated with gold and 1). The highest prevalence (9.61%) of metacercariae by examined under an FEG 250 SEM (FEI-Quanta, Hillsboro, month was observed in May 2016 in X. obvia, in April 2016 OR, USA). in Z. detrita, and in November 2016 in M. samsunensis Photomicrographs of the parasites were taken with a (Tables 2–4). Zeiss compound microscope and imaging system. Fixed unencysted Brachylaima sp. metacercariae Identification of the parasites was based on previously measure 700.2 ± 266.0 µm in length and 387.1 ± 134.7 published descriptions (Yamaguti, 1975; Butcher and µm in width. Metacercariae are leaf-shaped and possess 2 Grove, 2001; Barger and Hnida, 2008; Segade et al., 2011; suckers, oral and ventral. The oral sucker is slightly larger Valente et al., 2016). than the ventral sucker (Table 5). The oral sucker is located This study reports for the first time the natural at the anterior end of the body and the ventral sucker is occurrence of Brachylaima sp. unencysted metacercariae located on the first third part of the body. The oral and in the hepatopancreas of the land snails X. obvia, Z. detrita, ventral suckers are round to ovoid muscular disks, which and M. samsunensis. Brachylaima sp. metacercariae were can be seen relaxed or tightly contracted. The oral sucker detected in the hepatopancreas of 22 specimens (5.74%) is followed by a globular-shaped muscular pharynx. of the total 383 X. obvia specimens, 2 specimens (1.68%) The intestine is bifurcated after the pharynx, extending

Table 1. Total number of dissected snails by species, and their infection prevalence with Brachylaima sp. metacercariae.

Total number of dissected snails by species Number of infected snails Infection prevalence (%) 383 (Xerolenta obvia) 22 5.74 119 (Zebrina detrita) 2 1.68 121 (Monacha samsunensis) 21 17.35

Table 2. Prevalence of Xerolenta obvia infected with Brachylaima sp. metacercariae by month (spring and autumn).

Number of dissected snails by month Number of infected snails Infection prevalence (%) 0 (March 2016) - 0 46 (April 2016) 1 2.17 52 (May 2016) 5 9.61 0 (September 2016) - - 129 (October 2016) 2 1.55 156 (November 2016) 14 8.97

Table 3. Prevalence of Zebrina detrita infected with Brachylaima sp. metacercariae by month (spring).

Number of dissected snails by month Number of infected snails Infection prevalence (%) 0 (March 2016) - 0 56 (April 2016) 1 1.78 63 (May 2016) 1 1.58

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Table 4. Prevalence of Monacha samsunensis infected with Brachylaima sp. metacercariae by month (spring and autumn).

Number of dissected snails by month Number of infected snails Infection prevalence (%) 0 (March 2016) - 0 0 (April 2016) - 0 109 (May 2016) 18 16.5 0 (September 2016) - 0 0 (October 2016) - 0 12 (November 2016) 3 25

Table 5. Measurements and biometric data of the metacercariae of Brachylaima sp. (n: number of samples; SD: standard deviation; SE: standard error).

Metacercariae (n = 25) Measurements (µm) Range Mean SD SE Body length 238.5–1408.7 700.2 266.0 53.2 Body width 139.2–584.8 387.1 134.7 26.9 Oral sucker length 76.4–581.4 189.8 96.5 19.3 Oral sucker width 90.3–433.7 181.2 70.3 14.1 Ventral sucker length 60.2–326.8 142.2 50.9 10.2 Ventral sucker width 72.7–506.0 182.3 76.0 15.2

parallel to the lateral margins and ending blindly near the near the excretory and genital pores. Hair-like structures posterior end of the body. Food particles are visible in the encircle the oral and ventral suckers (Figures 1–3). intestine. Three gonads (2 testes and 1 ovary) are located To our knowledge, this study is the first record of close together at the posterior part of the body and near the natural occurrence of Brachylaima sp. metacercariae the posterior end of the body. The ovary is present between in the hepatopancreas of land snails. Some researchers the anterior and posterior testes. The uterus runs along have previously reported the presence of metacercariae the right side of the anterior testis, overpasses the ventral of Brachylaima spp. in the kidney, pericardial sac, body sucker, reaches up to the intestinal bifurcation, and then cavity near the female genital pore, and pedal glands of returns in front of the anterior testis. The genital pore is land snails (Mas-Coma and Montoliu, 1986; Thiengo and visible on the ventral surface and located at the anterior Amato, 1995; Butcher and Grove, 2001; Barger and Hnida, end of the anterior testis. The excretory pore is located at 2008; Barger, 2011; Segade et al., 2011; Valente et al., the posterior end of the body. The tegument has ladder- 2016; Nakao et al., 2017). The occurrence ofBrachylaima like elevations on the dorsal surface of the body, but it is sp. metacercariae in the hepatopancreas of mollusks is smooth on the ventral surface. When the metacercaria is surprising. Mollusks can acquire the cercariae through the tightly contracted, thin folds appear on the tegument and mouth, and the cercariae can migrate to the hepatopancreas small numerous holes become visible near the ventral from the intestine by the choledochal duct. sucker. On the anterior end of the body, at the level of No sporocysts and cercariae of Brachylaima sp. were the oral sucker, some dome-like papillae exist, which are detected in the hepatopancreas of mollusks in this study. presumed to possess sensory functions. The dome-like Brachylaima spp. use the mollusks as first intermediate papillae are also located in the oral and ventral suckers hosts. The first snail host is very typical, as brachylaimid and have no opening or cilium. No papillae are observed species use only one or a very small number of closely

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Figure 1. Photomicrographs of Brachylaima sp. metacercaria: a, b, c - relaxed; d - contracted and stained with Semichon’s acetocarmine. AT, Anterior testis; EP, excretory pore; GP, genital pore; I, intestine; P, pharynx; O, ovary; OS, oral sucker; PT, posterior testis; U, uterus; VS, ventral sucker. related mollusk species inhabiting the regional area. was based on the active times of land snails. They are in However, the second snail host appears to be less typical. hibernation during winter and become active in spring It generally belongs to another species living because of the warm weather and rain. Activated land in the same area as the first one. Sometimes it may even snails could acquire the cercariae of Brachylaima sp. from be other individuals of the first host species (Sirgel et al., other snails and also the egg of the parasite from definitive 2012). Although the relationship of coinfections within hosts; thus, the risk factor could be increased. In summer, one snail by the sporocysts, cercariae, and metacercariae land snails could be in an estivation period and might is complex, it can occur (Butcher and Grove, 2005; Nakao become active again in autumn because of warm weather. et al., 2017). Only one study has reported the occurrence Land snails with the infection are detected in spring and of Brachylaima sp. cercariae and sporocysts in a land snail may have acquired the parasite in spring or autumn of species, Helix aspersa, from Turkey (Köse at al., 2015). No the previous year. Similarly, land snails with the infection further studies on land snails as the intermediate hosts of detected in autumn may have acquired the parasite in the Brachylaima sp. have been reported in Turkey. Identifying previous autumn or spring. When the land snails become the species of Brachylaima from the larval stages living active, the larval stages continue to develop. in land snails is impossible. Further studies should be In the present study, the highest prevalence of the conducted on definitive hosts. parasite was observed in M. samsunensis (17.35%). This In the present study, the prevalence of infection by land species is endemic to Turkey and is a common species Brachylaima sp. metacercariae was high in X. obvia in May, in Kastamonu, whereas the other 2 snail species, X. obvia Z. detrita in April, and M. samsunensis in November. The and Z. detrita, have a wide distribution in Turkey and reason for selecting spring and autumn as the study period the central and eastern parts of Europe (Kebapçı, 2007;

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Figure 2. SEM images of the dorsal and ventral body surfaces of Brachylaima sp. metacercaria: a - from the ventral side; b - thin folds; c, d - elevations from the dorsal side. P, Dome-like papilla; OS, oral sucker; VS, ventral sucker; small numerous holes (arrowheads).

Figure 3. SEM images of the oral and ventral suckers of Brachylaima sp. metacercaria: a, b - oral sucker; c, d - ventral sucker. P, Dome- like papilla; hair-like structures (arrowheads).

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Audibert et al., 2010). Therefore, the infection can be easily Acknowledgment spread from the second intermediate hosts to the definitive We would like to express our appreciation to the Research host in places where these snail species exist. and Application Center of Kastamonu University for the In conclusion, further studies on several different land SEM images. snails as first and second intermediate hosts and definitive hosts will provide new knowledge about the life cycle and dissemination of Brachylaima spp.

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