Trichodinid fauna of Aphanius danfordii

ActA Acta Protozool. (2007) 46: 73–80 Protozoologica Trichodinid fauna of the toothcarp Aphanius danfordii (Boulenger, 1890) (Osteichthyes: Cyprinodontidae), an endemic ish from Sarıkum Lagoon Lake in Sinop (Turkey) Türkay ÖZTÜRK and Ahmet ÖZER Ondokuzmayıs University, Sinop Fisheries Faculty, Sinop, Turkey Summary. Ectoparasitic trichodinids of the toothcarp, Aphanius danfordii (Boulenger, 1890) were investigated during the period from May 2003 to April 2004 in ish collected from Sarıkum Lagoon Lake at Sinop, Turkey. A total of three trichodinid species including Trichodina domerguei Wallengren, 1897, Trichodina modesta Lom, 1970 and Tripartiella macrosoma Basson and Van As, 1987 were identiied on 423 ish specimens. Photomicrographs and morphometric data were presented for each species. The existence of three trichodinid species in relation to different length classes and the sex of toothcarp as well as their monthly occurences were also investigated and determined. This paper is the irst report on the trichodinid fauna present on toothcarp in Turkey. Trichodina modesta and Tripartiella macrosoma were recorded as new records for the parasite fauna in Turkey. Key words: ectoparasite, Aphanius danfordii, Trichodina domerguei, T. modesta, Tripartiella macrosoma. INTRODUCTION Trichodinids belong to one of the most commonly encountered parasitic ciliates. More than 200 species of trichodinid ciliophorans have been reported from the skin, ins, gills and urinary bladder of ishes. Despite the abundance of trichodinids, they have been little studied in Turkey. There are only a few studies on the parasites of the toothcarps, i.e. Coleman and Travis (1998) Hicks and Steele (2003). Address for correspondence: Türkay Öztürk, Ondokuzmayıs Üniversitesi, Sinop Su Ürünleri Fakültesi, 57000 Sinop, Türkiye, Fax: +90-368 287 6255, E-mail: turkay.ozturk@gmail.com The toothcarp, Aphanius danfordii (Boulenger, 1890) is a small cyprinodontid ish (7–8 cm maximum in total length), and inhabits brackish water of coastal lagoons, river-mouths and freshwater habitats (Demirsoy 1998). It is endemic to Sarıkum Lagoon Lake in Sinop, Turkey. The present paper deals with three ectoparasitic trichodinid species belonging to the genera Trichodina Ehrenberg, 1830 and Tripartiella Lom, 1959 collected from the toothcarp, A. danfordii. The existence of three trichodinid species in relation to different length classes and the sex of toothcarp as well as their monthly occurrences were also investigated. This paper is also the irst report on trichodinids of toothcarp from Sarıkum Lagoon Lake at Sinop, Turkey and elsewhere in the 74 T. Öztürk and A. Özer world. Trichodina modesta Lom, 1970 and Tripartiella macrosoma Basson and Van As, 1987 were recorded as new records for the parasite fauna in Turkey. MATERIALS AND METODS Specimens of A. danfordii were collected by net ishing in the estuary of the Sarıkum Lagoon Lake (42o 00′N; 34o 54′E), which is connected to the Black Sea. It is typically a lagoon and an eutrophic lake. The lake has a brackish characteristic with a salinity between 1–5‰. The total lake surface area is 184 ha with a watery area of 102 ha and a marshy area of 82 ha. The mean depth of the lake is approximately 1–1.6 m, with silt, muddy and sandy substrate. The lake is inhabited by some aquatic animals, such as turtles and snakes and eight ish species: toothcarp, Aphanius danfordii; lounder, Platichthys lesus; mullets, Mugil spp.; round goby, Neogobius melanostomus, three-spined stickleback, Gasterosteus aculeatus; sand smelt, Atherina boyeri and common carp, Cyprinus carpio. In addition, the lake area is also important for hundreds of aquatic bird species to rest, particularly during their winter migration. Sarıkum Lagoon Lake and the Sarıkum region have both been declared conservation areas by the Ministry of Forestry. Sampling was carried out on a monthly basis between the period May 2003 – April 2004. However, from October 2003 to March 2004 (5 months) no toothcarps were found in the sampling site. For parasitological examination, ish were transported alive in local water directly to the Sinop Fisheries Faculty Laboratory. A total of 423 ish specimens were investigated. The total lengths were measured and their sex determined at postmortem. Skin, ins and gills were examined under a ligth microscope, and scrapings of whole mucus from these parts of ish were taken on several slides. The total number of trichodinids was determined by screenning and counting the entire mucus material on each slide. Air dried smears were stained in accordance with the Klein’s silver nitrate (AgNO3) method (Lom and Dykova 1992) in order to study details of the adhesive disc. All morphological measurement were carried out by oil-immersion light microscopy (Nikon SE). All measurements are micrometres and follow the uniform speciic characteristics proposed by Lom (1958). In each case, maximum and minimum values were given, the arithmetic mean and standard error were followed in parentheses. In the case of radial pins, the mode was given instead of the arithmetic mean. The span of the denticle was measured from the tip of the blade to the tip of the ray. In the description of denticle elements, the format recommended by Van As and Basson (1989) was followed. The infestation prevalence (%) and mean intensity levels of the trichodinids were determined according to Bush et al. (1997). The prevalence and mean intensity values of Trichodina modesta, T. domerguei and Tripartiella macrosoma were given for pooled data rather than by each trichodinid species. Normal distribution of the data was tested by using Kolmogorov-Simirnow test. Kruskal-Wallis test (Nonparametric ANOVA) was performed to ind out the signiicant differences in the mean intensity values of trichodinids for infestation sites, length classes of ish as well as for the months in which this study was conducted. The difference between parasite loading on female and male ish were tested by the Mann-Whitney U-test. The analyses were carried out using the computer programmes GraphPad Instat 3.0 and SPSS 9.0. RESULTS During the present study three species of trichodinid ciliates were identiied. Aphanius danfordii was found to be infested with Trichodina modesta, Trichodina domerguei and Tripartiella macrosoma, the irst one being the most common species. The site of infestation of the three trichodinids on the toothcarp were different. Trichodina modesta was commonly found on the gills of the toothcarp, rarely on the skin and ins, for T. domerguei it was the reversed. Tripartiella macrosoma was, however, found only on the skin of the toothcarp. In addition, proportions of 100 : 20 : 1 (T. modesta : T. domerguei : Tripartiella macrosoma) were observed on the stained slides. Descriptions, photomicrographs and morphometrical data of the silver-impregnated specimens of all three species are presented below. Trichodina modesta Lom, 1970 (Figs 1A, 2A; Table 1) A medium-sized trichodinid with disc-shaped body. The centre of the adhesive dics of the specimens impregnated with silver nitrate is dark-stained. The blade of denticle is nearly sickle-shaped. The distal margin of blade is rounded and slants away from the border membrane. The tangent point is lat. The anterior margin of blade sharply curves down. The apex of blade is round, not touching y + 1 axis. Blade apophysis not visible. Posterior blade margin paralel to anterior blade margin. Blade connection thin. The central part of denticle narrow, pointed and extending to slightly more than halfway towards the y – 1 axis. The shape of section above and below the axis is similar. Ray connection short and thin. Rays slightly curved in posterior direction with tips extending slightly beyond y axes. Section of denticle above x axis to denticle below similar, ratio one. The morphometrical data are presented in Table 1. Trichodina domerguei Wallengren, 1897 (Figs. 1B, 2B; Table 1) A large trichodinid with disc-shaped body. The centre of the adhesive disc of the specimens impregnated with silver nitrate is clear with numerous dark granules. The sickle-shaped blade of denticle is broad illing large area between y axes. The distal margin of blade almost Trichodinids of the toothcarp Aphanius danfordii 75 Figs 1A, B, C. A – Trichodina modesta Lom, 1970; B – Trichodina domerguei Wallengren, 1897; C – Tripartiella macrosoma Basson and Van As, 1987. Specimens stained with silver-nitrate. Scale bar 10 µm. touches the border membrane. The apex of blade is round almost touching y + 1 axis. Tangent point round. Posterior blade margin fairly curved. Blade apophysis present, but not clearly visible. Blade connection thin. Central part well developed, but thin and long tapering to rounded point itting tigthly into preceding denticle. Ray connection short and thin. Base of ray thin, with ray bulbous towards broads and rounded point. Rays short and curved in posterior direction with tips extend- ing beyond y axes. Section of denticle above x axis to denticle below similar, ratio one. The morphometrical data are presented in Table 1. Tripartiella macrosoma Basson and Van As, 1987 (1C, 2C; Table 1) A small trichodinid with bell-shaped body. The adhesive disc cup-shaped. The border membrane is inely striated. Blade narrow, not curved. The apex of blade T. Öztürk and A. Özer 76 Fig. 2. Diagrammatic drawings of the denticles of trichodinids: A – Trichodina modesta Lom, 1970; B – Trichodina domerguei Wallengren, 1897; C – Tripartiella macrosoma Basson and Van As, 1987. Table 1. Morphometrical data and infestation sites of Trichodina modesta, T. domerguei and Tripartiella macrosoma (n: number of measured specimens) (range with arithmetic mean and standart error in parentheses) (all measurements in µm) from Aphanius danfordii. Species Trichodina modesta (n: 20) Trichodina domerguei (n: 20) Tripartiella macrosoma (n: 6) site gills, rarely skin and ins skin and ins, rarely gills only skin Diameter of 34–43 (38.4 ± 0.56) 27–36.5 (31.4 ± 0.62) 16.5–22.5 (19.6 ± 0.37) 60–76 (66.8 ± 2.87) 52–66 (58.0 ± 2.55) 34–44 (38.4 ± 1.97) 23.5–26.5 (24.7) 21–23 (22.0) 7–8 (7.5) 21–26 (23) 6–7 22–29 (27) 9–10 23–29 (28) 4 3.5–5 (4.3 ± 0.09) 3–4.7 (4.1 ± 0.12) 4–6 (4.8 ± 0.12) 6–8.2 (7.0 ± 0.35) 3.5–4.7 (4.3 ± 0.21) 9–11 (9.9 ± 0.33) 4–6 (5.0) 1.3–2 (1.6) 2 (2) body adhesive disc denticulate ring Number of denticle radial pins per denticle Length of blade ray denticle Span of denticle Width of central part border membrane 8.3–11.1 (10.1 ± 0.17) 1.2–2.5 (1.7 ± 0.07) 3–4.5 (3.6 ± 0.09) 12–16 (14.1 ± 0.64) 2.5–3 (2.8 ± 0.14) 4–5 (4.3 ± 0.19) 7–8.5 (7.6) 1 (1) 1–3 (1.98) Trichodinids of the toothcarp Aphanius danfordii very slightly curved. Tangent point round and lower than distal margin. The distal margin of blade rounded. Anterior blade margin slopes downwards. Two blade apophysis are present. The second apophysis pronounced, corresponding to indentation in anterior margin of next blade and sharply pointed. Posterior projection present and visible. Blade connection thin and long, of equal thickness throughout. The central part of denticle is rather small and delicate with ray extending directly from central part, almost in straight line with y axis. Ray is short and sharply pointed. Ratio of denticle above to below x axis more than two (2.5–2.7). The morphometrical data are presented in Table 1. Monthly prevalence and mean intensities of trichodinids infesting A. danfordii are reported in Table 2. The overall infestation prevalence (%) and mean intensity levels recorded from 423 ish specimens were 91.25% and 190.58 ± 28.27 trichodinids per infested ish, re- 77 spectively (Table 2). Both levels were also recorded for all body parts as well as for the sex and length clases of A. danfordii (Table 2). Statistically signiicant differences were determined in relation to the sex and length classes of ish and as shown in Tables 2 and 3. However, it must be noted that no statistically signiicant differences were determined between the infestation values of each of the six length classes of ish in all months, thus the data were pooled and analysed for six length classes without sampling months (Table 2). DISCUSSION Trichodinids are geographically a widely dispersed group of ectoparasites in freshwater, marine and euryhaline environments. Some trichodinids including Trichodina modesta parasiting Vimba vimba (Lom Table 2. Combined infestation prevalence (%) and mean intensity levels of three trichodinid species (T. domerguei, T. modesta and Tripartiella macrosoma) on Aphanius danfordii. n Prevalence (%) Mean intensity ± S.E. Months May 2003 June 2003 July 2003 August 2003 September 2003 March 2004 April 2004 Overall 62 71 51 65 67 52 55 100.0 85.9 100.0 80.0 83.6 98.1 96.1 423 91.3 Infestation site gills skin ins 423 423 423 86.1 68.1 35.7 Length classes of ish (mm) 34–37 38–41 42–45 46–49 50–53 54–57 * 15 49 175 129 47 8 66.7 85.7 92.0 94.6 93.6 87.5 Means followed by the same superscript letter are not signiicantly different. 56.3a ± 8.60 50.3a ± 5.66 519.2b ± 170.17 132.9ac ± 16.04 92.2a ± 15.56 71.7a ± 16.18 467.8bc ± 110.03 Statistical test used Kruskal-Wallis p < 0.05 Dunn’s 190.6 ± 28.87 577.80a ± 30.29 8.97b ± 0.57 5.99c ± 0.49 36.6 a ± 12.24 98.8 a ± 19.11 188.9 a ± 38.41 281.6 a ± 74.21 92.7 a ± 21.58 28.7 a ± 11.18 Kruskal-Wallis p < 0.05 Dunn’s Kruskal-Wallis p > 0.05 78 T. Öztürk and A. Özer Table 3. Monthly combined prevalence (P) and mean intensity (M.I.) values of three trichodinid infestations on female and males of Aphanius danfordii. Female Months May 2003 June 2003 July 2003 August 2003 September 2003 March 2004 April 2004 Overall n P (%) 36 39 25 37 38 36 29 240 100.0 84.6 100.0 83.8 78.4 97.2 100.0 91.3 Male M.I. ± S.E. 72.7 62.8 234.1 102.3 72.6 83.9 275.2 123.2 ± 12.39* ± 8.74* ± 52.81 ± 16.56* ± 16.66 ± 22.72 ± 61.48 ± 12.61 n P (%) 26 32 26 28 29 16 26 183 100.0 87.5 100.0 75.0 90.0 100.0 92.3 91.3 M.I. ± S.E. 33.6 35.6 815.7 178.1 113.3 44.9 700.6 279.0 ± 9.87* ± 5.79* ± 335.94 ± 29.08* ± 26.60 ± 12.41 ± 224.91 ± 63.89 * The differences in mean intensities between female and male ish in the same months are statistically signiicant. 1970b), Blicca bjoerkna (Wierzbicka 1997) and Abramis brama (Lom 1970b, Wierzbicka 1997, Gaze and Wooten 1998) and Tripartiella macrosoma parasiting Barbus eutaenia (Basson and Van As 1987) have been recorded in freshwater environments. This study is the irst that both trichodinid species are observed in a sligtly brackish environment. Trichodina domerguei is one of the most widely distributed trichodinids and it has been reported from variety of ish hosts living in freshwater, brackish and marine habitats (Lom and Stein 1966; Lom 1970a; Xu et al. 1999; Özer 2003a, b). In the present study, the morphometric data of the specimens of T. domerguei were larger in general than those reported from Enophrys bison by Lom (1970a), from Gasterosteus aculeatus and from Neogobius melanostomus by Özer (2003a, b), possibly relecting the differences of environmental factors and water chemistry as well as a result of host inluences. Trichodina modesta was the dominant species and found mainly on the gills, rarely on the skin and ins of the toothcarp. The present data are comparable with those reported by Lom (1970b), Arthur and Lom (1984), Basson and Van As (1994) and Wierzbicka (1997). These authors stated that this species was found only on gills. On the other hand, most of the skin specimens were T. domerguei in this study. Lom and Stein (1966) and Özer (2003a, b) also reported this species to be skin speciic and rarely found on the gills as well. Our indings on the morphological and morphometrical data variations of T. modesta and T. domerguei are also in agreement with the statements of the authors mentioned above. Only six Tripartiella macrosoma specimens were found in stained slides throughout the investigation period. The morphometrical data and denticle form of Tripartiella macrosoma correspond well with the original description by Basson and Van As (1987) where this species was found on Barbus eutaenia, a small endemic cyprinid of Southern Africa. Our data on the infestation site of T. macrosoma are different from that reported by Basson and Van As (1987) who found this species only on the gills. It must be noted that the number of T. macrosoma individuals determined here is very low and they were all encountered on the skin. It is very well known that Tripartiella species are all, without exception, gill parasites. The difference in our inding on different location on ish could be the exceptional presence of this parasite on the skin itself or its presence on only one out of 423 ish specimens. We can easily exclude the possibility of cross infestation to be caused by ish being small-sized as we are conident that we conducted a carefull examination process throughout the study period. Trichodinids often show seasonal changes in prevalence and intensity of infestation and occurrence of trichodinids is generally related to the rise in water temperature. Some authors reported peak levels of trichodinid infestation in spring and early summer (Özer and Erdem 1998; Özer 2000; 2003a, b). The highest mean intensity levels in the present study were 519.22 ± 170 and 467.83 ± 110.03 trichodinids per infested ish in Trichodinids of the toothcarp Aphanius danfordii July 2003 (early summer) and April 2004 (spring) respectively (Table 2). But, following the highest intensity of infestation occurred in July 2003, there was a sharp decrease in August 2003. This may be attributed to the presence of more uninfested young ish in August 2003 in the sampling area. While T. modesta and T. domerguei were observed throughout the study period, T. macrosoma was only recorded in July, possibly due to the different temperature needs of each species. No statistically signiicant differences were determined between the mean intensity values of different ish length classes. The size of toothcarp wasn’t a factor affecting the number of trichodinids here. On the other hand, some authors noted an increased tendency in the mean intensity of Trichodina spp. in relation to the length of ish (Özer and Erdem 1998; Özer 2003a, b) as a result of the difference in host species which were also different in their sizes. In addition, it is known that as the ish gets longer, the space for parasite settlement increases. This was true in the present study up to the length class of 46–49 mm and there was a decrease in the following length classes. This might be a possible result of more longer ish had more developed immunological response to infestation. The number of studies on the existence of trichodinid parasites on female and male ish is very rare and almost no statistically signiicant differences in their existence was found (Özer 2000). No statistically signiicant difference in the overall mean intensity values between both sexes of ish found in the present study. However, some statistically signiicant differences in May 2003, June 2003 and August 2003 were determined, possibly because of the spawning activity of the toothcarps that further stress caused by maturation of eggs which caused deppression on ish. Pickering (1977), Pickering and Christie (1980) and Urawa (1992) attributed infestation differences in different sex of ish to several factors such as rhythmical changes in epidermis of host ish, a decrease in number of AB-positive mucous cells and an increase in PAS-positive mucous cell. Further stress on ish in July 2003 when ish started their spawning activity might have occurred. Similar observations were also done in April 2004 when examined ish had mature eggs that corresponded a sharp increase in the trichodinid number encountered on ish. In conclucion, this paper is the irst research study conducted on the existence of the trichodinid fauna of Aphanius danfordii. 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