01_Kutsokon_05_21.indd UDC UDC 597.551.2(282.247.32) AGE AND GROWTH OF THE EUROPEAN BITTERLING, RHODEUS AMARUS (CYPRINIDAE, ACTINOPTERYGII), IN THE UDAY AND PEREVOD RIVERS (DNIPRO BASIN, UKRAINE) A. Podobailo1, A. Shukh1, Yu. Kutsokon2* 1Taras Shevchenko National University of Kyiv, Volodymyrska st., 64/13, Kyiv, 01030 Ukraine 2Schmalhausen Institute of Zoology NAS of Ukraine, vul. B. Khmelnytskogo,15, Kyiv, 01030 Ukraine *Corresponding author E-mail: carassius1@ukr.net Yu. Kutsokon (https://orcid.org/0000-0001-9721-5638) Age and Growth of the European Bitterling, Rhodeus amarus (Cyprinidae, Actinopterygii), in the Uday and Perevod Rivers (Dnipro Basin, Ukraine). Podobailo, A., Shukh, A., Kutsokon, Yu. — European bitterling age and growth were examined in 2017, 2018, and 2019 in the Uday and Perevod rivers in Pyriatynsky National Park (Poltava Region, Ukraine). Th e population was represented by fi ve age groups (0+ to 4+); specimen age was determined by counting annuli on scales. Th e annuli were visible and oft en displayed as irregularities in circuli in the centrolateral part of the scales. Standard specimen length varied from 14 to 51 mm; weight varied from 0.09 to 2.5 g. Th e length-weight relationship was W = 0.00005*SL2.78; W{ = 0.00048*SL 2.18; W} = 0.00028*SL 2.32 for both sexes, males and females, respectively. Th e sex ratio was 1 : 1.26. Th e most abundant group in the samples from the Uday were young-of-the- years, and 1+ fi sh in the samples from the Perevod. As for mature specimens, 3+ fi sh predominated in both samples. Th e fastest relative growth was during the second year of life. K e y w o r d s : bitterling, scale, length-weight relationship. Introduction Bitterling is a fi sh of the Cyprinidae family, which is the only shell-dwelling species in the overall ichthyofauna of Ukraine, including the Uday River (Koshovyy & Podobaylo, 2017). Females lay eggs in the mantle cavity of bivalves (Anodonta anatina (L.) and A. cygnea (L.), Unio pictorum (L.), U. tumidus (Philipsson) (Reichard et al., 2006) and U. crassus (Philipsson) (Tatoj et al., 2017) using an ovipositor. Th e lifespan of bitterling is fi ve years, but the majority doesn’t survive past the fi rst year (Kottelat and Freyhof, 2007). Previously, studies on bitterling growth were conducted in Poland (Przybylski and Garsia-Berthou, 2004), Greece (Koutrakis et al., 2003), and Turkey (Tarkan et al., 2005). Th e population consists of fi ve age groups (the oldest age group is 4+). Th ere is also data on the populations of this species in the Tym and Elbe rivers (Zhul’kov & Nikiforov, 1987; Holčík, 1960) where the maximum age was 6+ and 8+, respectively. Bitterling reach a length of 40 % or 50 % of their maximum length during their fi rst year of life (Przybylski and Garsia-Berthou, 2004). Zoodiversity, 55(5):361–368, 2021 DOI 10.15407/zoo2021.05.361 Ecology 362 A. Podobailo, A. Shukh, Yu. Kutsokon Th e European bitterling is found mainly in the additional system of rivers, fl oodplains, lakes, and ponds. It prefers habitats with slow-fl owing or stagnant water, small depths, and sandy, rocky or muck bottoms. However, it can also occur in rapids (Przybylski, Ziêba, 2000; Reichard et al., 2002). Bitterling schools are usually found among macrophytes near the bottom and rarely go out into open water. Bitterlings inhabit European freshwater basins of the Black and Caspian seas. In Ukraine, they can be found in all large rivers, except for Crimean waters (Movchan & Smirnov, 1983). Bitterling is the most abundant species in the water bodies of Pyriatynsky National Park (Glotova et al., 2012; Koshovyy et al., 2018). It is listed in the Berne Convention on the Conservation of European Wildlife and Natural Habitats (1979) and is protected by Council Directive 92/43/EEC on the conservation of natural habitats and wild fauna and fl ora. In 2016, Pyriatynsky National Park has become a part of the Emerald Network (UA0000077). Research of key species pecularities, including those of bitterling, is actively performed for preservation. Th e aim of this study is to describe the age, growth, and population structure of bitterling in the protected water system of the Uday and Perevod rivers in Pyriatynsky National Park, in order to contribute to an understanding of the environmental and geographical variation on bitterling life history characteristics, and to fi ll the gaps in the management of this species. Material and methods Pyriatynsky National Park is a nature protection territory in the Pyriatyn District of Poltava Region, located in the valleys of Uday, Perevod, and Ruda rivers. Uday is a left bank second-order tributary of the Dnipro. It is 327 km long, with 62 km fl owing through the territory of the national park. Perevod is the largest right bank tributary of the Uday. It is 68 km long, of which 27 km fl ow through the national park. Sampling was done during daytime at two permanent hydrobiological monitoring stations (table 1) using a 6 m long beach seine with a 5 mm mesh. Th e fi rst station was located in Perevod near the village of Sasynivka close to a railway bridge (50°18.487 N, 32°26.032 E). Th e second station was located on a beach of the Uday in the village of Keibalivka (50°18.345 N, 32°30.093 E) (fi g. 1). Fig. 1. Study locations: 1 — Sasynivka; 2 — Keibalivka. T a b l e 1 . Investigated bitterling specimens from the Uday and Perevod rivers River Date of sampling Immature Males Females Total Uday 1.08.18 73 20 17 110 27.07.19 2 20 20 42 total 75 40 37 152 Perevod River 22.07.17 0 15 7 22 31.07.18 7 12 9 28 26.07.19 53 5 4 62 total 60 32 20 112 363Age and Growth of the European Bitterling Rhodeus amarus in the Uday and Perevod Rivers… Th e scales were prepared for age determination according to the classical method (Chugunova, 1959): ten scales from above the lateral line under the dorsal fi n were taken from each fi sh; specimen standard length (SL) was measured with a sliding gauge to the nearest 0.1 mm; weight was determined to the nearest 0.01 g using electronic scales; sex was determined visually by the appearance of gonads and presence of the ovipositor in females if it presents Slides with scales for microscopy were prepared in the laboratory. Th e scales were soaked in a diluted ammonia solution (3 %), and each scale was separated with needles. Th ey were cleaned of epithelial residues and placed between two slides pressed tightly together. ID, weight, and length of each specimen were indicated on a label. Aft erward, the slides were left to dry out (Chugunova, 1959) and later examined using a stereomicroscope МБС-10 and photographed using a digital camera SIGETA UCMOS05100KPA-U-NA-N-C-SQ-NA and a lens adapter for the microscope FMA050. Th e total radius of the scales and radii of the annuli were measured with an accuracy of 0.01 px using a ToupTek ToupView 3.7.2270. Measurements were performed from the scale centre to the outer edge of the annuli in the place with the largest diameter. Growth was back calculated using Lea’s method (Chugunova, 1959): Sn/S = Ln/L, where S is the total radius of the scale (px), Sn is the radius of the annuli n, L is total fi sh length (mm), and Bertalanff y’s method (Ricker, 1975): Lt = Linf (1 – exp(–k (t–t0))), where Lt is fi sh length at age t (years), Linf, k, t0 are coeffi cients. Growth rate was calculated using the formula of instantaneous growth rate (IGR) (Ricker, 1975). IGR was back-calculated according to Lea’s method: IGR = lgLt–1 — lgLt, where Lt–1 is fi sh length corresponding to the length of the scales at the end of the last year of life, Lt is fi sh length corresponding to the length of the scales at the beginning of the last year of life. Length-weight relationship (Ricker, 1975): W = a∙SLb, where W is weight (g), SL — standart length (mm), a and b — coeffi cients. Th e Shapiro-Wilk test was used to check the distribution for normality. Student’s t-test, χ² test, ANOVA, ANCOVA were used for statistical processing of the obtained results. Student’s test and ANOVA, ANCOVA were used to compare morphological features. Th e χ ² test was used to assess the accuracy of the sex ratio (Lakin, 1990). Statistical processing was performed in STATISTICA 7. Results Th e Sasynivka station was covered with dense beds of higher aquatic vegetation including Lemna minor L., Spirodela polyrhiza (L.), Sagittaria sagittifolia L., Elodea canadensis Michx. Th e river bottom at the sampling point was composed of gravel. Th e Keibalivka station was also covered with dense beds of higher aquatic vegetation represented by Lemma minor, Spirodela polyrhiza, Ceratophyllum demersum L., Nuphar lutea L. (Smith). Th e river bottom at the sampling site was sandy. Th e beach was a popular local recreation area. It was also actively grazed by geese. Th e studied bitterling population in the Uday and Perevod rivers consisted of fi ve age groups (0+ to 4+). A total of 264 specimens was investigated. Annuli were best seen on the back and side parts of the scales. Th e shape of the scales changed with age: in juvenile bitterlings it was more rounded, and acquired the shape of an ellipse as the fi sh grew (fi g. 2). Due to these changes, the measured radius did not have a constant place but moved along the caudal part of the scales with age. Standard length varied from 14 to 51 mm, weight from 0.09 to 2.5 g. Length-weight Fig. 2. Bitterling scales. Th e arrows show the annuli. 364 A. Podobailo, A. Shukh, Yu. Kutsokon relationship was W = 0.00005*SL2.78; W{ = 0.00048*SL 2.18; W} = 0.00028*SL2 .32 (r2 = 0.94, df = 262, t = –5.08, r2 = 0.63, df = 70, t = –4.07, r2 = 0.68, df = 55, t = –3.18 for both sexes, males, and females, respectively). Th e length-weight relationship is said to be isometric, there is cubic relationship between length and weight. Sex ratio in both samples was 57 females and 72 males or 1 : 1.26. No statistically signifi cant deviation from the ratio 1: 1 was detected (χ2 = 0.013, р = 0.05). ANCOVA showed no statistically signifi cant diff erence between the length of males and females from diff erent rivers (F = 0.7, p = 0.05, F = 2.1, p = 0.05 for males and females respectively). However, this diff erence was present in immature specimens: bitterlings from the Perevod were larger than specimens from the Uday (F = 18.8, p = 0.05) (table 2). Th is may be because the samples were taken in diff erent years. Annual ichthyofauna monitoring in the waters of the Pyriatynsky National Park, revealed that bitterling was the most common and abundant species in the Uday and its tributaries (Koshovyy et al., 2015). Th e share of bitterling in ichthyological samples varied from 18.8 % in 2016 to 73.9 % in 2015. Only in the catches of 2016 the relative number of bitterling was inferior to the Rutilus rutilus (L.). In 2016, there was an abrupt decline in the number of bitterling; almost no juvenile specimens were observed in samples. In 2017, a large number of juveniles appeared, and almost no age-1+ were observed in samples. Th e generation gap gradually narrowed in 2018–19 and almost disappeared in 2019 (fi g. 3). T a b l e 2 . Th e average length (SL) of bitterling depending on sex River Immature Males Females mean SD range Mean SD range Mean SD range Uday 19.83 2.86 14–26 39.26 4.22 32–51 39.74 3.51 33–47 Perevod 24.56 2.53 19–29 38.73 4.65 30–48.5 38.1 4.78 29–47.2 Fig. 3. Length weight ratio of bitterling in samples from the rivers Uday and Perevod in 2016–2019. 365Age and Growth of the European Bitterling Rhodeus amarus in the Uday and Perevod Rivers… All lengths and age groups were represented in the specimens selected for the study because samples were collected during 2017–2019. Th e most represented group from the Uday samples was 0+, while 1+ dominated in samples from the Perevod. Among sexually mature specimens, 3+ predominated in both samples (table 3). Th e average specimen length in each age group was less than the back calculated length. However, empirical average lengths of males and females at age 3+ and 4+ did not diff er from each other), but in the age 2+ females are larger than males (ANOVA post-hoc test, 6 groups, p = 0.002). Th e parameters of the von Bertalanff y growth equation showed further variation in growth rates between sexes (table 4). Th e diff erence between sexes can be explained by using the largest fi sh in group as asymptotic SL. Th e growth rate of bitterling slowed down with age (table 5). During the second year of life, the relative length gain of fi sh was highest for both sexes. During the second and third years of life, bitterling from the Perevod and Uday rivers grew at the same rate, but during the fourth year of life, both males and females from the Uday grew slower (p = 0.0027 for the relative length gain and p = 0.0024 for instantaneous growth rate). Ranking of fi sh by weight and length depending on age was performed for each age group. Th e minimum and maximum weights and lengths were determined for each age; for immature T a b l e 3 . Mean observed and back–calculated total lengths (mm) obtained from measurement of scales in rivers Uday and Perevod combined data Age group Standard length Number of sp. Length at the age of, mm 1+ 2+ 3+ 4+ 0+ 20.16 ± 2.61 93 1+ 25.86 ± 2.01 42 Males 2+ 35.00 ± 3.04 25 16.07 34.02 3+ 39.33 ± 1.95 30 13.23 30.24 38.75 4+ 44.41 ± 2.93 17 14.65 32.13 40.16 43.94 Females 2+ 34.87 ± 2.66 18 19.94 36.08 3+ 39.40 ± 1.64 24 16.14 31.80 39.40 4+ 43.98 ± 1.87 15 18.99 35.13 41.30 45.10 T a b l e 4 . Estimates of Linf, k and t0 of female and male bitterling in the Uday and Perevod rivers Parameters Females Males Sex combined n 57 72 129 Linf 48.5 51 51 k 0.636 0.439 0.449 t0 1.01 0.58 0.54 T a b l e 5 . Relative length gains and instantaneous growth rate of bitterling in the Uday and Perevod rivers Parameters Ages 1–2 2–3 3–4 Uday River Relative length gain, % 33.71 ± 6.40 24.48 ± 4.82 16.85 ± 4.52 Instantaneous growth rate 0.18 ± 0.04 0.12 ± 0.03 0.08 ± 0.02 Perevod River Relative length gain, % 33.52 ± 5.55 25.43 ± 4.93 22.05 ± 4.38 Instantaneous growth rate 0.18 ± 0.4 0.13 ± 0.03 0.11 ± 0.02 366 A. Podobailo, A. Shukh, Yu. Kutsokon 0+ and 1+ fi sh, the lengths almost did not overlap, while weights overlapped almost completely. When bitterlings became mature (age-1+), their growth rate accelerated as well as weight gain, so a clear boundary of 30 mm between 1+ and 2+ was observed for both sexes (fi g. 4). Th e linear growth of mature bitterling was quite slow. Fish length ranges partially overlapped. In contrast to linear growth, bitterling weight varied greatly within one age group, so it is impossible to determine the age group of fi sh belongs according to its weight. Discussion Determination of bitterling age has been the subject of several studies (Holčík, 1999; Tarkan, 2005; Koutrakis, 2003), but only Przybylski (2004) confi rmed the age determined by scale reading using operculum. Annual rings on the operculum were clearly visible, and the number of annual rings on scales and operculum coincided. Determination of bitterling age using operculum is as accurate as using scales. Bitterlings from the Uday and Perevod rivers were characterized by a short life cycle. We found fi ve age groups (the oldest was 4+). Th is coincided with the general pattern observed in other studies (Holčík, 1960; Koutrakis, 2003; Przybylski and Garsia-Berthou, 2004; Tarkan, 2005). On the other hand, the results were very diff erent from the results from the Elbe River (Zhul’kov & Nikiforov, 1987; Wagler, 1949 cited by Holčík, 1999), where the oldest age group was group 8+. Smith et al. (2000) determined that the sex ratio in the bitterling population in oxbow lakes in the Czech Republic was always 1 : 1. Fig. 4. Length and weight of bitterling depending on age. 367Age and Growth of the European Bitterling Rhodeus amarus in the Uday and Perevod Rivers… In the Rihios River (Greece) (Koutrakis, 2003), males outnumbered females. Th e sex ratios may diff er in diff erent populations and in diff erent years.  Th e reasons behind this variation may be diff erent and numerous: species diff erences, fi sh body length, seasonal aspects, feeding and reproductive periods, diff erent growth rates in males and females, earlier maturation of one of the sexes, overall mortality and accidental (due to fi shing technique) mortality diff erence for each sex and perhaps environmental conditions (Peczalska, 1968). In the Uday river the sex ratio is 1 : 1.08 and in the Perevod river the sex ratio is 1 : 1.6, with the number of males dominating over females but near to 1 : 1 for both rivers. In the population of Lake Ömerli reservoir in Istanbul (Tarkan, 2005), on the contrary, females dominate: the sex ratio is 1 : 2.32. In bitterling, as in other cyprinids, there is some sexual dimorphism in average length of males and females (Holčík, 1960), but it was absent in the Uday and Perevod rivers. As suggested by Reichard & Jurajda (1999), Tarkan (2005) stated that bitterling reached sexual maturity in both sexes at age-1+ at a length of 30–35 mm. We found that maturity in bitterling occurred at a length of about 30 mm in the third year of life. All specimen with a length of 30 mm (2+) were sexually mature. Among specimens shorter than 30 mm long, there was only one female of 29 mm long (2+), the others were immature 0+ and 1+. Probably such a diff erence was because fi sh in the Uday and Perevod rivers reached a shorter length than in other water bodies. Th us, the maximum specimen length in both the Uday and the Perevod was 51 mm, and the average length ± SD was 37.1 ± 5.9 mm. Th ese values are much lower than those given for fi sh from other water bodies of Ukraine (e. g., average length for the Dnipro basin was 52.4 ± 0.1 mm, for the Danube basin — 52 ± 0.2 mm (Movchan, 1983)), and than values obtained in other studies. Th is may be due to conditions in the Uday and the Perevod. Th ese rivers have a more northern location compared to other studied water. Th us, later maturity was most likely associated with smaller bitterlings. We noted fl uctuations in the number of bitterlings in the catches during 2014–2019. During the summer of 2016, there was an extremely unfavorable oxygen regime in the Uday (Koshovyy & Podobaylo, 2017) that probably led to mass death of the young-of-the- year, which were absent in the catches in 2016. Bitterling is a slow-growing species. During the fi rst year of life, fi sh reach only 40  % of their maximum possible length. Similar data was found on bitterling from the Ömerli (Tarkan, 2005). Other studies (reviewed by Holčík 1999; Koutrakis, 2003) indicate that bitterling reached 50 % or more of their maximum length during the fi rst year of life. Although the Wieprz-Krzna population (Przybylski and Garsia-Berthou, 2004) is the northernmost of the studied bitterling populations, fi sh from there are larger than from the more southern Uday. Th is diff erence may be due to the geographical location and related diff erences in water temperature and food supply, as has been studied for other small fi sh (Mann et al., 1984). In addition, we determined the limits of body length of diff erent age groups in the population of bitterling in the Uday and Perevod rivers, which was not done in previous studies. With this, we can approximately determine the age of bitterling according to their length. It is quite diffi cult to distinguish between the young-of-the-year and age-1+ fi sh based only on their lengths. Th is is possible for mature specimen. According to the graph (fi g. 4), there is a clear boundary between age-1+ and age-2+ fi sh, which was 30 mm. Older age groups can be distinguished roughly: most likely, a fi sh of 30–35 mm long is 2+, while bitterling longer than 40–42 mm is more likely to be 4+. It is most diffi cult to distinguish age-2+ and age-3+ fi sh based on length, because their lengths overlap the most. Bitterling is the most abundant of 32 fi sh species in the Pyriatynsky National Park (Koshovyy et al., 2018). 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