Natural infection of the land snails Xerolenta obvia, Zebrina detrita, andMonacha 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
Brachylaimidae Joyeux & Foley, 1930 is a large family of digenean trematodes comprising several genera and species with a worldwide distribution. The genus Brachylaima Dujardin, 1843 is a member of this family and comprises the primary endoparasites of endothermic vertebrates, including birds, mammals, and humans (Yamaguti, 1975; Guisantes et al., 1994; Butcher and Grove, 2001, 2005; Lunaschi and Drago, 2012; Reda and El-Shabasy, 2016; Suleman and Khan, 2016). Land snails and slugs are required as the first and second intermediate hosts in the life cycle of Brachylaima spp. (Yamaguti, 1975; Butcher and Grove, 2005; Barger and Hnida, 2008; Barger, 2011; Sirgel et al., 2012; Valente et al., 2016). First intermediate hosts have sporocysts and cercariae, and second intermediate hosts have metacercariae of Brachylaima spp. Infection occurs when the definitive hosts consume raw mollusks containing metacercariae (Butcher and Grove, 2001, 2005; González-Moreno and Gracenea, 2006; Barger and Hnida, 2008; Barger, 2011; Sirgel et al., 2012; Gracenea and Gállego, 2017; Nakao et al., 2017).
Although some studies have been conducted on the second intermediate host mollusk species of Brachylaima spp. worldwide (Thiengo and Amato, 1995; Barger and Hnida, 2008; Barger, 2011; Sirgel et al., 2012; Valente et al., 2016; Gracenea and Gállego, 2017; Nakao et al., 2017), only sporocysts and cercariae have been reported in Helix aspersa as a first intermediate host in Turkey (Köse et al., 2015); metacercariae have not been reported.
The goal of this study was to investigate the prevalence and morphological features of infection of the land snails Xerolenta obvia, Zebrina detrita, and Monacha samsunensis (Mollusca: Pulmonata) by the metacercariae of Brachylaima sp. (Trematoda: Brachylaimidae) in Kastamonu, Turkey.
The land snails X. obvia, Z. detrita, and M. samsunensis were picked up following rainfall from areas in Kastamonu Province in April and May (spring) and October and November (autumn) in 2016. A total of 383 specimens of X. obvia, 119 specimens of Z. detrita, and 121 specimens of M. samsunensis were picked up. In the laboratory, the land snails were dissected while alive and their hepatopancreases were taken out. These organs were spread on clean glass slides with 0.6% NaCl solution. Using a mounted needle, the hepatopancreas was incised and a thin film of the liquid was dropped onto a slide for investigation of parasites under a light microscope. After the metacercariae of Brachylaima sp. were identified, they were fixed in 5% formalin. The fixed parasites were coated with an adhesive mixture and stained with Semichon’s acetocarmine.
Some samples were prepared for scanning electron microscope (SEM) examinations, for which the formalin-fixed specimens were washed in distilled water overnight and postfixed in 2% (w/v) osmium tetroxide aqueous solution for 4 h at room temperature. After 5 washes with distilled water, the specimens were dehydrated through
Abstract: This study investigated the prevalence of infection, and some of its morphological features, of the land snails Xerolenta obvia, Zebrina detrita, and Monacha samsunensis (Mollusca: Pulmonata) by metacercariae of Brachylaima sp. (Trematoda: Brachylaimidae) 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.
Received: 29.05.2017 Accepted/Published Online: 23.10.2017 Final Version: 10.01.2018
Short Communication
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an ethanol series, kept for 20 min in each concentration, and dried in a CO2 critical-point dryer (Imai et al., 1992). The dried specimens were sputter-coated with gold and examined under an FEG 250 SEM (FEI-Quanta, Hillsboro, OR, USA).
Photomicrographs of the parasites were taken with a Zeiss compound microscope and imaging system.
Identification of the parasites was based on previously published descriptions (Yamaguti, 1975; Butcher and Grove, 2001; Barger and Hnida, 2008; Segade et al., 2011; Valente et al., 2016).
This study reports for the first time the natural occurrence of Brachylaima sp. unencysted metacercariae in the hepatopancreas of the land snails X. obvia, Z. detrita, and M. samsunensis. Brachylaima sp. metacercariae were detected in the hepatopancreas of 22 specimens (5.74%) of the total 383 X. obvia specimens, 2 specimens (1.68%)
of the total 119 Z. detrita specimens, and 21 specimens (17.35%) of the total 121 M. samsunensis specimens (Table 1). The highest prevalence (9.61%) of metacercariae by month was observed in May 2016 in X. obvia, in April 2016 in Z. detrita, and in November 2016 in M. samsunensis (Tables 2–4).
Fixed unencysted Brachylaima sp. metacercariae measure 700.2 ± 266.0 µm in length and 387.1 ± 134.7 µm in width. Metacercariae are leaf-shaped and possess 2 suckers, oral and ventral. The oral sucker is slightly larger than the ventral sucker (Table 5). The oral sucker is located at the anterior end of the body and the ventral sucker is located on the first third part of the body. The oral and ventral suckers are round to ovoid muscular disks, which can be seen relaxed or tightly contracted. The oral sucker is followed by a globular-shaped muscular pharynx. 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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parallel to the lateral margins and ending blindly near the posterior end of the body. Food particles are visible in the intestine. Three gonads (2 testes and 1 ovary) are located close together at the posterior part of the body and near the posterior end of the body. The ovary is present between the anterior and posterior testes. The uterus runs along the right side of the anterior testis, overpasses the ventral sucker, reaches up to the intestinal bifurcation, and then returns in front of the anterior testis. The genital pore is visible on the ventral surface and located at the anterior end of the anterior testis. The excretory pore is located at the posterior end of the body. The tegument has ladder-like elevations on the dorsal surface of the body, but it is smooth on the ventral surface. When the metacercaria is tightly contracted, thin folds appear on the tegument and small numerous holes become visible near the ventral sucker. On the anterior end of the body, at the level of the oral sucker, some dome-like papillae exist, which are presumed to possess sensory functions. The dome-like papillae are also located in the oral and ventral suckers and have no opening or cilium. No papillae are observed
near the excretory and genital pores. Hair-like structures encircle the oral and ventral suckers (Figures 1–3).
To our knowledge, this study is the first record of the natural occurrence of Brachylaima sp. metacercariae in the hepatopancreas of land snails. Some researchers have previously reported the presence of metacercariae of Brachylaima spp. in the kidney, pericardial sac, body cavity near the female genital pore, and pedal glands of land snails (Mas-Coma and Montoliu, 1986; Thiengo and Amato, 1995; Butcher and Grove, 2001; Barger and Hnida, 2008; Barger, 2011; Segade et al., 2011; Valente et al., 2016; Nakao et al., 2017). The occurrence of Brachylaima sp. metacercariae in the hepatopancreas of mollusks is surprising. Mollusks can acquire the cercariae through the mouth, and the cercariae can migrate to the hepatopancreas from the intestine by the choledochal duct.
No sporocysts and cercariae of Brachylaima sp. were detected in the hepatopancreas of mollusks in this study. Brachylaima spp. use the mollusks as first intermediate hosts. The first snail host is very typical, as brachylaimid species use only one or a very small number of closely
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).
Measurements (µm)Metacercariae (n = 25)
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
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related mollusk species inhabiting the regional area. However, the second snail host appears to be less typical. It generally belongs to another land snail species living in the same area as the first one. Sometimes it may even be other individuals of the first host species (Sirgel et al., 2012). Although the relationship of coinfections within one snail by the sporocysts, cercariae, and metacercariae is complex, it can occur (Butcher and Grove, 2005; Nakao et al., 2017). Only one study has reported the occurrence of Brachylaima sp. cercariae and sporocysts in a land snail species, Helix aspersa, from Turkey (Köse at al., 2015). No further studies on land snails as the intermediate hosts of Brachylaima sp. have been reported in Turkey. Identifying the species of Brachylaima from the larval stages living in land snails is impossible. Further studies should be conducted on definitive hosts.
In the present study, the prevalence of infection by Brachylaima sp. metacercariae was high in X. obvia in May, Z. detrita in April, and M. samsunensis in November. The reason for selecting spring and autumn as the study period
was based on the active times of land snails. They are in hibernation during winter and become active in spring because of the warm weather and rain. Activated land snails could acquire the cercariae of Brachylaima sp. from other snails and also the egg of the parasite from definitive hosts; thus, the risk factor could be increased. In summer, land snails could be in an estivation period and might become active again in autumn because of warm weather. Land snails with the infection are detected in spring and may have acquired the parasite in spring or autumn of the previous year. Similarly, land snails with the infection detected in autumn may have acquired the parasite in the previous autumn or spring. When the land snails become active, the larval stages continue to develop.
In the present study, the highest prevalence of the parasite was observed in M. samsunensis (17.35%). This land species is endemic to Turkey and is a common species in Kastamonu, whereas the other 2 snail species, X. obvia and Z. detrita, have a wide distribution in Turkey and the central and eastern parts of Europe (Kebapçı, 2007;
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.
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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 spread from the second intermediate hosts to the definitive host in places where these snail species exist.
In conclusion, further studies on several different land snails as first and second intermediate hosts and definitive hosts will provide new knowledge about the life cycle and dissemination of Brachylaima spp.
AcknowledgmentWe would like to express our appreciation to the Research and Application Center of Kastamonu University for the SEM images.
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