Abstract 1 INTRODUCTION RESEARCH ARTICLE

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1 Received: 19 June 216 Revised: 13 March 217 Accepted: 1 May 217 DOI: 1.12/aqc.2798 RESEARCH ARTICLE Crayfish in central and southern Ukraine with special focus on populations of indigenous crayfish Astacus pachypus (Rathke, 1837) and their conservation needs Tomas Policar 1 Volodymyr Bondarenko 1 Oles Bezusyj 2 Vlastimil Stejskal 1 Jiri Kristan 1 Oleksandr Malinovskyi 1 Aiman Imentai 1 Miroslav Blecha 1 Yuriy Pylypenko 3 1 Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceske Budejovice, Vodnany, Czech Republic 2 Institute of Fisheries of the National Academy of Agrarian Sciences of Ukraine, Kiev, Ukraine 3 Faculty of Fisheries and Nature, Kherson State Agricultural University, Kherson, Ukraine Correspondence Tomas Policar, University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zatisi 728/II, Vodnany, Czech Republic. policar@frov.jcu.cz Funding information Ministry of Education, Youth and Sports of the Czech Republic, Grant/Award Number: CENAKVA (No. CZ.1.5/2.1./1.24), CENAKVA II (No. LO125 under the NPU I program) and GAJU 6/216/Z Abstract 1. The thick clawed crayfish (Astacus pachypus Rathke, 1837) is the least studied indigenous crayfish species in Europe. Information about its distribution and biology is out of date by more than 15 years. 2. This study identified 94 localities with potential occurrence of thick clawed crayfish in eight southern and central regions of Ukraine, using questionnaire and literature analysis. Based on the information obtained, a field survey was conducted to examine and confirm the current distribution and abundance of crayfish species and evaluate basic water quality and habitat characteristics in each locality. 3. Details of density, sex ratio in the catches, health and moulting condition, threat level and water quality were identified for each population of A. pachypus. 4. Only four populations of this species were found, in lower parts of the Dnieper River, cooccurring with Astacus leptodactylus Eschscholtz, in Kakhovka reservoir near Vesele village, two sites on the Dnieper River near Nova Kakhovka town and near Prydniprovske village, and one locality on the Dnieper s tributary the Ingulec River near Sadove village. 5. Populations of thick clawed crayfish at three sites had low crayfish densities of.3.4 crayfish m 2 or catch efficiency.2 crayfish per trap night. Only one locality on the Dnieper River, close to Nova Khakovka, had a stronger population with higher density (1.7 crayfish m 2 ). 6. Healthy thick clawed crayfish inhabit larger water bodies with stable environments and good water quality. Female catch per unit effort was lower, and they had a higher percentage of chelae injuries compared with males. 7. All of the identified thick clawed crayfish populations are exploited by uncontrolled fishing for consumption and there is an urgent need for conservation of both the crayfish and their habitat. KEYWORDS conservation, distribution, Dnieper River, East Europe, habitat, thick clawed crayfish, Ukraine 1 INTRODUCTION The thick clawed crayfish (Astacus pachypus Rathke, 1837) is considered to be the least widespread native crayfish in Europe today (Gherardi & Souty Grosset, 21). Within Eastern Europe, this mesohaline indigenous crayfish is known from both freshwater and brackish localities in Ukraine and Russia (Cherkashina, 1999; Fűreder et al., 26; Starobogatov, 1995). Freshwater populations were recorded in the basins of the rivers Dnieper, South Bug, Don and Volga. In addition, thick clawed crayfish were known to occupy Ukrainian and 6 Copyright 217 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/aqc Aquatic Conserv: Mar Freshw Ecosyst. 218;28:6 16.

2 POLICAR ET AL. 7 Russian coastlines of the Black Sea and Azov Sea (Cherkashina, 1999; Holdich et al., 26; Kouba, Petrusek, & Kozák, 214; Skurdal & Taugbøl, 22). However, field information from the last three decades of the last century (Brodsky, 1981, 1983; Cherkashina, 1974, 1975, 1976, 1999; Sokolsky, Ushivtsev, Mikouiza, & Kalmikov, 1999; Starobogatov, 1995), and current updates are lacking in most cases. Some theoretical updates on the occurrence of thick clawed crayfish, with an expressed need to confirm these updates, were published in Kouba et al. (214). These authors mentioned a reduction of the current range of A. pachypus in the eastern part of the Black Sea, in the Crimean Peninsula and in the north eastern Azov Sea including the Don River. Mezhzherin, Kostyuk, Garbar, Zhalai, and Kutishchev (215) briefly noted the occurrence of one small population of thickclawed crayfish in the lower parts of the Dnieper River near the villages of Ponyatovka ( N, E) and Novotyaginka ( N, E) in the Kherson region of Ukraine, but without details on current population status and habitat conditions. Three indigenous crayfish species narrow clawed crayfish (Astacus leptodactylus Eschscholtz, 1823), noble crayfish (Astacus astacus Linnaeus, 1758) and thick clawed crayfish occur in current Ukraine (Kouba, Buřič, & Petrusek, 215; Kouba et al., 214). Narrow clawed crayfish are widespread, abundant and commercially harvested for food, and their conservation status there has not yet been established (Fűreder et al., 26). Noble crayfish occur only in the western part of Ukraine, including water bodies of the Carpathians and the Podolsk highlands with declining trends (Mezhzherin et al., 215) and this species is considered vulnerable (Dovgal, 29). The thick clawed crayfish is not covered by Ukrainian fishery and nature conservation rules and legislation, which are not well established there for practical nature conservation (Movchan, 215). However, this species is currently identified as a species in danger of extinction in the whole of Europe (Gherardi & Souty Grosset, 21). Recently two localities with non native marbled crayfish (Procambarus fallax Hagen, 187 f. virginalis) were recorded in Ukraine near the cities of Odessa and Dnepropetrovsk. The second locality close to the Dnieper River is especially important as it would allow its further expansion which could adversely affect all populations of native crayfish in this river and catchment (Novitsky & Son, 216). The thick clawed crayfish is the least studied indigenous crayfish in Europe. Most information relating to their distribution, reproduction and ecology is out of date and repeated from publication to publication, because of a lack of research focused on this species (Gherardi & Souty Grosset, 21; Holdich et al., 26; Kouba et al., 215). The aim of this study was to identify and summarize localities with potential occurrence of thick clawed crayfish in Ukraine, which were then monitored to update occurrence data for this crayfish, its condition, habitat, threat level and conservation needs. 2 METHODS 2.1 Localities with potential occurrence of thick clawed crayfish The first stage of this study, at the end of 211, was literature analysis that enabled a detailed review of information about the occurrence of thick clawed crayfish in Ukraine to be compiled. After identifying potential sites of their occurrence, a questionnaire providing a morphological key for determining this crayfish species (Holdich et al., 26; Pöckl, Holdich, & Pennerstorfer, 26) together with the potential localities was sent to the following Ukrainian colleagues: Dr S. Tarasjuk (Institute of Fisheries of the National Academy of Agrarian Sciences of Ukraine, Kiev), Dr I. Sherman (Kherson State Agricultural University Faculty of Fish Farming and Ecology, Kherson), Dr O. Voloshkevych (National Academy of Sciences of Ukraine Ukrainian Danube Delta Biosphere Reserve, Vilkovo), Dr V. Kostyuk (Zhytomyr Ivan Franko State University, Zhytomyr), Dr I. Dykukha (State Agency of Fisheries of Ukraine Novokakhovskyj State Institute, Obryvka) and Dr V. Plugatarev (State Agency of Fisheries of Ukraine State Sturgeon Institute, Dniprovske). These partners were considered to be the most important key specialists for aquatic fauna in Ukraine and likely to have information about the present occurrence of thick clawed crayfish, as well as close contact with the Ukrainian Hunting and Fishing Association ( the State Ecological Inspectorate of Ukraine ( and active fishermen. Over a 1 year partnership, 35 questionnaires were sent to 287 individual fishermen, fishery or aquatic researchers and nature conservationists. All addressees were asked about potential localities for thick clawed crayfish. After the collection and analysis of answers, a list of potential localities for thick clawed crayfish occurrence in eight Ukrainian regions (Figure 1; Table 1 and Table S1 S6, Supporting Information) was created between February and April Field monitoring of crayfish occurrence During August and September 213, when crayfish in Ukraine are most active, a field expedition took place to selected regions (Figure 1) with the aim of visiting and exploring all identified sites with potential occurrence of thick clawed crayfish. Monitoring crayfish occurrence (with the main emphasis on adult crayfish with TL 6 mm) in all sites followed protocols used in other field studies (Kozak, Duris, & Policar, 22; Kozák, Policar, & Ďuriš, 24; Policar & Kozák, 25). Initially, all localities were divided into three broad types based on water volume: (1) small and shallow brooks and rivers, (2) large, deep rivers, bays and, (3) ponds, lakes and (Ďuriš, Drozd, Horká, Kozák, & Policar, 26). Different methods for monitoring or capture were used in different types of sites. In the first category, a manual survey of refuges (stones, shoreline burrows, littoral vegetation and its roots) was carried out by two men using hand nets to search three similar suitable sites in the river bed with an area of 3m 2 (for a total bottom area of 9 m 2 ) for 3 minutes. For each locality with crayfish presence, catch efficiency (catch per unit effort CPUE) of adult crayfish (TL 6 mm) was determined as the average number of crayfish caught (± SD) per man hour, and crayfish density as average number of crayfish caught per square metre. Crayfish occurrence in deeper and larger rivers, bays and was investigated by diving (two men searched three areas of the bottom, each approximately 6 m 2 with a total area of 18 m 2 for each locality for 1 hour, and the catch efficiency and crayfish density were calculated as before. Most localities were examined in the morning (5. 9.) or evening ( ) when crayfish were most

3 8 POLICAR ET AL. FIGURE 1 Map of Ukraine showing regions numbered and colour coded (1 Cherkassy, 2 Kirovohrad, 3 Dnipropetrovsk, 4 Zaporizhia 5 Crimean Peninsula, 6 Kherson, 7 Mykolaiv, 8 Odessa), where potential localities for thick clawed crayfish (A. pachypus) occurrence were identified and field surveys carried out TABLE 1 Summarized data from all sites surveyed in eight Ukrainian regions identified as potential localities for thick clawed crayfish (A. pachypus A.p.) with the percentage of suitable localities for European native crayfish and confirmed crayfish occurrence (A.l. A. leptodactylus), including CPUE (1 Number of crayfish caught per man hour evaluated in brooks, rivers, bays and ; 2 Number of crayfish caught per trap night evaluated in ponds, lakes and ) and crayfish density (3 Number of crayfish caught per m 2 evaluated in brooks, rivers, bays and ). Data are expressed as means ± standard deviations, n means number of localities of each water body in each region. Different superscript letters in the same column indicate significant differences in CPUE and crayfish density among regions and types of water body Region Cherkassy Kirovohrad Dnipropetrovsk Zaporizhia Crimean Peninsula Kherson Mykolaiv Odessa Type of water body Brooks and small rivers n Suitable localities (%) Crayfish present (%) Crayfish species CPUE (1) (2) A.l b ± A.l. 2. b ± Density (3) A.l. 4. b ±.47.4 a ± A.l. 3.2 b ± A.l. 2.4 b ± a ± A.l. 6. c ±.4.7 b ± A.l. 2. b ±.2 a ± A.l. 2.4 b ± A.l d ± d ± A.p. 7.3 c ± b ± A.l. 3.4 b ±.85 1 A.p..2 a ± A.l. 5.4 e ± c ± A.l f ± d ± 1.9

4 POLICAR ET AL. 9 active. In the third category of localities (ponds, lakes and ), Fritid plastic traps (58 mm 24 mm 2 mm) from Finland were used to monitor crayfish occurrence. Ten traps baited with fresh muscle of common carp (Cyprinus carpio) were installed on the bottom during the evening and left overnight. Traps and any crayfish caught were checked and collected the next morning. Catch efficiency was determined as the average number of adult crayfish (± SD) caught per trap night. The exact crayfish density per square metre of the bottom was not calculated for any monitored pond, lake or reservoir using this method of crayfish capture. Each crayfish caught was identified to species based on previous experience and the publications by Brodsky (1981), Holdich et al. (26) and Pöckl et al. (26). 2.3 Determination of water quality and general habitat character of visited localities Basic water quality parameters such as oxygen saturation (%), water temperature ( C) (measured on the bottom in small shallow brooks, or 5 mm below the water surface in ponds,, deeper and large rivers etc.), ph, salinity ( ) and conductivity (ms m 1 ) were measured with a Multiline P4 oxymeter (WTW measuring and analytical equipment, Czech Republic Ltd) and Combo ph tester EC HI (Hanna instruments, Czech Republic Ltd) in each locality visited. Other detailed water quality parameters such as NH 4 N, NO 3 N, NO 2 N, N total, PO 3 4 P, P total, COD Mn (chemical oxygen demand, BOD 5 (biochemical oxygen demand) were also analysed and measured at each site where thick clawed crayfish occurrence was confirmed. All water samples (volume 2 L) were transported in plastic vials to the Laboratory of Aquatic Toxicology and Ichthyopathology of the Faculty of Fisheries and Protection of Waters, University of South Bohemia in the Czech Republic within 36 hours and the water quality parameters were determined there. After measuring, all water samples were safely disposed of (Horáková, Lischke, & Grünwald, 1989). General habitat characteristics including benthic substrate, leaf litter and shelter availability (e.g. burrow, cobble, tree roots, littoral macrophyte vegetation) were evaluated at each site following Kozak et al. (22). Any visible organic pollution from agricultural activities or human settlements, and erosion or habitat alteration and fragmentation were recorded at all sites. All these parameters were assessed with the aim of defining suitability of each locality for European native crayfish occurrence. 2.5 Threat level to thick clawed crayfish populations After identifying and surveying sites with thick clawed crayfish, each site was observed over 24 hours to get an indication of levels of exploitation and harvesting of crayfish by local residents. At each site, the number of active crayfishermen was recorded by observation. Each fisherman was contacted and questioned about his fishing permit and catch (numbers of crayfish caught, percentage of thick clawed crayfish and the sex and size proportion in the catch). The interviews were conducted with technical help from the local Kherson state fishing inspectorate. The information was supplemented by information on thick clawed crayfish offered for sale at the local fish market at Pyatachok (N , E ) which was close to all localities with verified populations of thick clawed crayfish. All crayfish sold were checked, identified and the proportion of each species was recorded. The results were used to define the exploitation pressure on thick clawed crayfish, the level of threat, and the requirements for conservation. 2.6 Statistical analysis All data are presented as means (± SD). Statistical analysis was performed using Statistica software 6.1 (StatSoft, Inc. Czech Republic). Before analysis, data for CPUE, crayfish density, TL and W were checked for normality (Kolmogorov Smirnov test) and homoskedasticity of variance (Bartlett s test). One way analysis of variance (ANOVA) was applied to determine differences in TL and W between females and males of A. pachypus, and in the crayfish density among all sites examined. Two way analysis of variance (ANOVA) was used (species and method of crayfish capture) to determine differences in CPUE. When a significant difference was detected (P <.5), Dunnett s multiple range test was applied during this study. Principal component analysis (PCA) was used to investigate the influence of all measured environmental variables on crayfish distribution across the study area. Environmental variables (oxygen saturation, water temperature, ph, salinity and conductivity) were used as continuous variables, whereas crayfish presence was used as a discrete factor. The environmental dataset used was not transformed in this analysis. 3 RESULTS 2.4 Thick clawed crayfish condition As well as crayfish CPUE, density and sex ratio, crayfish individual total length (TL) and body weight (W) were determined using a tape with 1 mm increments and an electronic scale Mettler (model AE 2) with an accuracy.1 g, following Policar et al. (211). Moult phase determined following Reynolds (22), crayfish health condition (evidence of disease, ectoparasites or commensals on the body surface) following Kozubíková Balcarová and Horká (215) and chelae injuries following Kouba, Buřič, Policar, and Kozák (211) were recorded for all thickclawed crayfish captured in the survey and those caught by crayfishermen and confiscated by the local Kherson state fishing inspectorate. 3.1 Crayfish occurrence and density The detailed information obtained from 94 sites is given in Table 1 and Tables S1 6 (Supporting Information). Crayfish were found at 61 sites (64.9%). Narrow clawed crayfish were the main crayfish species in all localities except one where thick clawed crayfish were dominant. Astacus leptodactylus abundance or CPUE fluctuated from.2 11 crayfish per m 2 or crayfish per man hour and crayfish per trap night. The lowest density and CPUE was found in rivers in the Zaporizhia region, an irrigation channel and two local ponds in the Crimean Peninsula. The highest density and CPUE were identified in Dnistrovskyj Bay near Maiaky in the Odessa region and in Kakhovka reservoir near Novooleksandrivka in the Kherson region

5 1 POLICAR ET AL. (Table S1 6, Supporting Information). Narrow clawed crayfish inhabited mainly deep and large rivers (n = 19), (n = 17) and bays (n = 17). The least inhabited localities for this crayfish species were brooks (n = 1) and (n = 1). It did not occur in ponds, lakes and, or in rivers, bays and of the Zaporizhia region and its occurrence was low also in brooks and small rivers of the Crimean Peninsula (Table 1). Thick clawed crayfish were observed in only four sites (4.3%), all in the Kherson region (Table 1 and Table S4, Supporting Information) of the lower reaches of the Dnieper River: Kakhovka reservoir near Vesele village ( N E), two sites on the main stream of the Dnieper River near Nova Kakhovka town ( N E) and near Prydniprovske village ( N E), and at one site on a major tributary of the Dnieper River the Ingulec River near Sadove village ( N E). In all these localities, thick clawed crayfish co occurred with narrow clawed crayfish. Densities and CPUE of thick clawed crayfish ranged from crayfish per m 2 or 3 15 crayfish per man hour and.2 crayfish per trap night. In three localities the density or CPUE of co occurring narrow clawed crayfish was higher than that of thick clawed crayfish. However, one thick clawed crayfish population in the main stream of the Dnieper River near Nova Kakhovka town ( N E) had a density of 1.7 crayfish per m 2 or CPUE 15 crayfish per man hour, higher than that of co occurring narrow clawed crayfish (Table S4, Supporting Information). 3.2 Water quality and habitat characteristics at sites with crayfish Summarized site information for each locality, its basic water quality parameters and suitability for occurrence of European native crayfish are given in Table 1 and Tables S1 S6 (Supporting Information). In total, 88.3% (n = 83) of localities examined were classified as suitable for native crayfish. Some 22 localities were without observed crayfish but would probably be suitable for native crayfish. In total, 11.7% (n = 11) of the localities in this study were identified as unsuitable for native crayfish for the following reasons: organic pollution (n = 9; 81.8%) which included visible organic pollution from agricultural activities or human settlements and low temperatures (n = 2; 18.1%) which did not exceed 15 C and 17 C (Tables S2, S3, Supporting Information). The least suitable localities were in the Zaporizhia region in ponds, lakes or and rivers, bays and and in the Crimean Peninsula, in brooks and small rivers (Table 1). Sites with narrow clawed crayfish ranged from 5 to 391 m above mean sea level (mamsl). Water quality varied, with oxygen saturation from 83 to 23%, water temperature C, ph , salinity.1 to >2. and conductivity 281 to >3999 ms m 1. Thick clawed crayfish were caught only in larger water bodies at low altitude: Kakhovka reservoir ( 12 mamsl), the Dnieper (2 8 masmsl) and the Ingulec rivers ( 3 mamsl), with a narrow range of basic water quality parameters including oxygen saturation %; water temperature C; ph ; salinity.2.3 and conductivity ms m 1 (Tables S1 S6, Supporting Information). This was confirmed with a PCA showing 62.7% cumulative variability of environmental data, with a wide spectrum of conditions for A. leptodactylus compared with A. pachypus which prefer localities with a narrower range of ph, oxygen level, salinity and conductivity (Figure 2). Most thick clawed crayfish were caught in depths of 3 4 m, where the bottom was characterized by 4% sand, grain size of 5 7 mm, 25% clay with burrows, 25% rock and stones, and 1% pebbles 3 45 mm. More detailed water analyses in localities with A. pachypus confirmed good water quality with a narrow range of the following values: NH 4 N mg L 1,NO 3 N.8.15 mg L 1,NO 2 N.5.7 mg L 1,N total mg L 1,PO 3 4 P.8.16 mg L 1, P total mg L 1, COD Mn mg L 1 and BOD mg L 1 (Table 2). FIGURE 2 Principal component analysis (PCA) showing the relationship between environmental variables and the distribution of the crayfish Astacus leptodactylus (A.l.) and Astacus pachypus (A.p.) from 61 sites in eight Ukrainian regions

6 POLICAR ET AL. 11 TABLE 2 Water quality variables in four localities with confirmed thick clawed crayfish (A. pachypus) occurrence in the Kherson region of Ukraine Locality Parameter Nova Kakhovka Prydniprovske Sadove Ingulec River Vesele Kakhovka reservoir Oxygen saturation (%) Water temperature ( C) ph Salinity ( ) Conductivity (ms m 1 ) NH 4 N (mg L 1 ) NO 3 N (mg L 1 ) NO 2 N (mg L 1 ) N total (mg L 1 ) PO 3 4 P (mg L 1 ) P total (mg L 1 ) COD Mn (mg L 1 ) BOD 5 (mg L 1 ) Condition of thick clawed crayfish In total, 46 adult thick clawed crayfish were caught and measured at four sites. Information on A. pachypus sex and size proportion, chelae injury and moult phase from these sites is given in Table 3. The highest number of these crayfish (n = 3) was taken in the Dnieper River near Nova Kakhovka. Lower numbers were caught in the Dnieper River near Prydniprovske (n = 8) and the Ingulec River (n = 6), with the lowest (n = 2) in Kakhovka reservoir near Vesele village. Sex ratios at all sites showed more males than females: in the Dnieper River near Nova Kakhovka, 6.%; the Dnieper River near Prydniprovske, 62.5%; the Ingulec River, 66.7% and Kakhovka reservoir near Vesele, 1%. Lower numbers of females caught were mainly the result of their lower activity caused by preecdysis and postecdysis moult phases. Total lengths of males ranged from 89.5 ± 11.7 to 92.3 ± 13.6 mm and weights from 34.2 ± 15.8 to 35.1 ± 16.9 g. Females caught in three localities had lower total lengths, from 76.3 ± 8.4 to 79.3 ± 9.3 mm and weights from 15.8 ± 5.1 to 17.8 ± 6.2 g. All 29 males caught from all localities were in the interecdysis moult phase, having a clean and hardening carapace. In total, eight females and one female from two sites in the Dnieper River were in postecdysis with a soft and clean carapace. In all localities, either one or two females were in preecdysis (with a soft, old carapace) or interecdysis (with a hard old carapace). This indicates that mid September was the moulting TABLE 3 Sex and size proportion, injured chelae (%) and moult phase of thick clawed crayfish catches in four localities in the Kherson region of Ukraine. Biometric data are expressed as means ± standard deviations. Different superscript letters in the same column indicate significant differences between female and male TL (individual total length) or W (individual body weight) Locality Parameter Nova Kakhovka Prydniprovske Sadove Ingulec River Vesele Kakhovka reservoir Total catch (no. of individuals) Males % (no. of individuals) 6 (18) 62.5 (5) 66.7 (4) 1 (2) TL (mm) 92.3 ± 13.6 b 9.8 ± 12.8 b 89.5 ± 11.7 b 9.2 ± 12.7 W (mm) 35.1 ± 16.9 b 34.8 ± 16.3 b 34.4 ± 15.8 b 34.2 ± 15.8 Injured chelae (%) Moult phase postecdysis (no. of individuals) preecdysis (no of individuals) interecdysis (no. of individuals) 18 Females % (no. of individuals) 4 (12) 37.5 (3) 33.3 (2) TL (mm) 79.3 ± 9.3 a 77.8 ± 8.7 a 76.3 ± 8.4 a W (mm) 17.8 ± 6.2 a 16.1 ± 6. a 15.8 ± 5.1 a Injured chelae (%) Moult phase postecdysis (no. of individuals) preecdysis (no. of individuals) interecdysis (no. of individuals)

7 12 POLICAR ET AL. period of female thick clawed crayfish in the localities visited. From a detailed examination to establish crayfish health, there were no visible diseases, ectoparasites or commensals. A higher percentage of damaged chelae were found in females compared with males (Table 3). 3.4 Illegal and unregulated exploitation of thickclawed crayfish populations Observations on exploitation of thick clawed crayfish are summarized in Table 4. The most common fishing method was homemade plastic traps (an average of 12 traps per crayfisherman) used in three localities except for the Dnieper River near Nova Kakhovka. Here, where the strongest population of A. pachypus was found, six crayfishermen used an unknown number of netting traps with net wings. This fishing method was involved in the heaviest exploitation, with a total of 375 crayfish caught (327 individuals of A. lepdodactylus and 48 of A. pachypus, including 46 males and two females). The total catch from other localities ranged from 35 (the Ingulec River near Sadove: 34 individuals of A. leptodactylus and one male of A. pachypyus) to 7 crayfish (Kakhovka reservoir near Vesele: 64 individuals of A. leptodactylus and six males of A. pachypus). The proportion of narrow clawed crayfish ranged from 87.2% (the Dnieper River near Nova Kakhovka) to 97.1% (the Ingulec River near Sadove). Thick clawed crayfish accounted for only % of all catches (Table 4). Generally, the size of the crayfish was not representative of the population because crayfishermen selected the largest crayfish, mainly males (95.8 1%), for market and food. However, it was significant that all crayfishermen caught crayfish without any permits or local regulations, indicating that all fishing was completely illegal. Ukrainian crayfishermen did not recognize that the A. pachypus they caught is a separate and endangered species compared with narrow clawed crayfish. All crayfish caught were prepared for sale in a local fish market at Pyatachok and for consumption. Crayfish from all catches were confiscated and stocked back to each locality. During a visit to a local fish market, live crayfish offered by six different dealers were checked. In total, 2456 live marketable crayfish were reviewed and only three dead and four live thick clawed crayfish (in total.3%) were found there. Again, there was no understanding that the thick clawed crayfish is a different species to narrow clawed crayfish, and the use of thick clawed crayfish for consumption was confirmed as common practice. 4 DISCUSSION 4.1 Current occurrence of thick clawed crayfish in Ukraine In the identification of localities in Ukraine potentially holding thickclawed crayfish, most attention was focused on southern Ukrainian regions on the coast of the Black and Azov Seas (Holdich et al., 26; Kouba et al., 214). The results of the questionnaire indicated that this species occurs in all southern regions except the Donetsk region. The questionnaire also showed the potential presence of thick clawed crayfish in central Ukraine (Cherkassy, Kirovohrad, Dnipropetrovsk regions). This accords with previous information from Holdich (22) which suggested that A. pachypus may occur in Kremenchuk and Dniprodzerzynsk and the Dnieper River of central Ukraine. However, after 2 months of intensive field survey involving 94 localities in eight Ukrainian regions, A. pachypus was found at only four localities within the Kherson region, thus confirming a very restricted presence of A. pachypus in Ukraine. Pöckl (21) published a similar conclusion after his expedition in the Mykolaiv region during 28. The present occurrence of A. pachypus is concentrated in the lower reaches of the Dnieper River in the Kherson region, contrary to information in Holdich (22), Holdich et al. (26) and Kouba et al. (214). Low densities or catch efficiencies of adult crayfish (.3.4 crayfish per m 2 or.2 crayfish per trap night or 3 4 crayfish TABLE 4 1 day exploitation by local fishermen of thick and narrow clawed crayfish populations in four localities in the Kherson region of Ukraine, with detailed information about the sex and size proportion of A. pachypus Locality Parameter Nova Kakhovka Prydniprovske Sadove Ingulec River Vesele Kakhovka reservoir Number of fishermen Fishing method Net traps Plastic traps Plastic traps Plastic traps Total catch (individuals) Portion of Astacus leptodactylus % 87.2 (327) 96. (48) 97.1 (34) 9. (64) (no. of individuals) Portion of Astacus pachypus % 12.8 (48) 4. (2) 2.9 (1) 8.6 (6) (no. of individuals) Males % (no. of individuals) 95.8 (46) 1 (2) 1 (1) 1 (6) TL (mm) ± ± ± 19.4 W (g) 18.8 ± ± ± 11.9 Females % (no. of individuals) 4.2 (2) TL (mm) 89.7 ± 8.6 W (g) 26.4 ± 3.4 TL individual total length; W individual body weight

8 POLICAR ET AL. 13 per man hour) were found at three sites. These recorded densities for Ukrainian thick clawed crayfish are different from those given by Gherardi and Souty Grosset (21) for the north west coast of the Caspian Sea (.2.8 or 1 crayfish per m 2 ) and Cherkashina (1999) for the Eastern Caspian Sea (.5.1 crayfish per m 2 ) or the Don River near Starocherkassk (.1 crayfish per m 2 ), respectively. Very few (n = 1) adult thick clawed crayfish were caught by Mezhzherin et al. (215) in the Dnieper River near Ponyatovka and Novotyaginka villages. These places are 1 to 2 km from sites identified in this study near Prydniprovske and Sadove villages and 5 to 6 km from the site near Vesele village. This information suggests that thick clawed crayfish may be distributed across the whole stretch of the Dnieper River between Kakhovka reservoir and Prydniprovske village. This study also found a larger population of A. pachypus (with a density of 1.7 crayfish per m 2 or CPUE 15 per man hour) in the stretch of the Dnieper River near Nova Kakhovka town about 5 m below the dam at Kakhovka reservoir. This locality, where a total of 3 caught and 48 confiscated adult crayfish of A. pachypus were examined for the purposes of this study, contains a highly valuable thick clawed crayfish population. This population will be studied in future research on morphology, genetics, reproduction, aquaculture, behaviour and ecology as recommended by Holdich (22), Holdich et al. (26), Kouba et al. (214) and Mezhzherin et al. (215) and applied to the future conservation of this species in Ukraine. Crayfish monitoring and capture, especially in the main stream of the Dnieper River or in, was complicated because of the size and depth of the water bodies and high flow rates. For example, the Kakhovka reservoir near Vesele village is m wide, with an average depth of 2 m. The main stream of the Dnieper River, where thick clawed crayfish were caught, had an average flow rate of about 26 m 3 s 1 and a width of m. These conditions could have reduced the efficiency of crayfish capture and did not allow juvenile monitoring, so it is possible that relatively higher crayfish densities may exist in some of the survey sites. 4.2 Thick clawed crayfish in other countries The documented populations of thick clawed crayfish in Ukraine provide much needed updates on the present distribution of this crayfish species in Europe. Besides Ukraine, this crayfish species is said to occur within Europe in Russia (the Don and the Volga rivers, coast of Azov and Caspian Sea; Cherkashina, 1999; Gherardi & Souty Grosset, 21; Holdich et al., 26). Outside Europe, thick clawed crayfish inhabit shallow coastal waters of the Caspian Sea in Azerbaijan, Kazakhstan and Turkmenistan (Cherkashina, 1999). Kouba et al. (214) mentioned the occurrence of A. pachypus on the Russian and Azerbaijani western coast of the Caspian Sea. However, any speculations about the present distribution of A. pachypus in the Caspian Sea (Holdich et al., 26; Kouba et al., 214) need to be supported by field investigations which are lacking over the last 15 or more years. Other information related to the occurrence of thick clawed crayfish in Moldova and Romania in Holdich et al. (26) and Kouba et al. (214) has not been confirmed by any field surveys. Similarly, information about its occurrence in the Bulgarian Devnya River (Bulgurkov, 1964; Holdich et al., 26) is also unconfirmed (Todorov et al., 214). Hubenova, Vasileva, and Zaikov (21) published a note stating that A. pachypus does not occur in Bulgaria, and the first record of this species by Bulgurkov (1964) was an erroneous determination. 4.3 Thick clawed crayfish conditions during the monitoring in Ukraine Only 46 caught and 57 confiscated adult thick clawed crayfish were obtained at all sites and investigated in this study. However, this is currently the highest published catch of this species in Europe. Total lengths and weights and different sizes of both sexes tallied with size ranges of thick clawed crayfish given in Cherkashina (1999), Gherardi and Souty Grosset (21), Holdich et al. (26) and Pöckl et al. (26). Fewer females than males were caught in all localities. The sex ratio in catches changes throughout the year and depends on several factors including moulting, mating and carrying offspring (Policar & Kozák, 25). The time of sampling was probably a factor, as most females caught were in pre and post moult phases of ecdysis and had soft carapaces and lower activity compared with all males caught which had hard carapaces after moulting and were probably at the highest level of feeding activity before autumn (Policar & Kozák, 25; Reynolds, 22). The greater frequency of injuries of chelae found in females than in males was probably related to male aggressive behaviour and predation by other animals. 4.4 Ukrainian localities with both thick and narrow clawed crayfish Co occurrence of thick and narrow clawed crayfish was evident in all Ukrainian localities where thick clawed crayfish were confirmed. This has not been noted in any scientific literature. This study showed that these crayfish species live together in one locality. However, narrowclawed crayfish were the main species in three localities, with infrequent occurrence of thick clawed crayfish. This may be related to the higher pleopodal fecundity (2 4 eggs versus 27 5 eggs) and ecological plasticity of narrow compared with thick clawed crayfish (Holdich et al., 26; Kozák et al., 211; Reynolds, 22; Skurdal & Taugbøl, 22). However, one locality (in the Dnieper River near Nova Kakhovka) had higher densities of thick compared with narrowclawed crayfish. During crayfish monitoring at this locality, a specific site with a total area of approximately 75 m 2 was identified where the density of A. pachypus was highest. The exact boundaries of the thick clawed crayfish population were observed, with higher densities of narrow clawed crayfish in surrounding areas. This thick clawed crayfish population structure was confirmed during a 1 day observation of the locality and discussion with local crayfishermen and the Kherson state fishing inspectorate. Information about aggressive behaviour of thick clawed crayfish towards narrow clawed crayfish, and the occupation and protection of deeper and stony localities by thick clawed crayfish against other crayfish, was also mentioned during this expert discussion. This study found scattered occurrences of thick clawed crayfish in other localities, but several other thick clawed crayfish populations could have been overlooked during crayfish monitoring of the main stream of the Dnieper River. Mosaic distribution of thick clawed crayfish and limited occurrence of this crayfish species

9 14 POLICAR ET AL. should be studied by a future field survey to examine the Dnieper River from Nova Kakhovka to Kherson town and provide better biological and ecological characterization, including the migratory ability of this crayfish. 4.5 Typical habitat of thick clawed crayfish in Ukraine In this study, thick clawed crayfish were found only in larger and deeper water bodies (Kakhovka reservoir, the Dnieper and the Ingulec rivers) providing a stable aquatic environment, narrow water quality range, sufficient natural sand clay stone pebble substrate, adequate shelter and aquatic and littoral vegetation (Mezhzherin et al., 215) compared with shallow, locally polluted, summer drying or cold brooks, smaller rivers and ponds. Preference of this crayfish species for deep localities reported by Cherkashina (1999), Gherardi and Souty Grosset (21) and Holdich et al. (26) was confirmed in this study. At the same time, the narrow range of good water quality seems to be a very important factor supporting the occurrence of A. pachypus. However, tolerance of thick clawed crayfish to higher water salinity (Cherkashina, 1999; Fűreder et al., 26) was not found during this study (Figure 2). 4.6 Threats to thick clawed crayfish populations The present study examined preselected localities where crayfish were observed in the past by Ukrainians. However, 11.7% of these localities were assessed as unsuitable for native crayfish mainly owing to their organic contamination from agricultural activities or human settlements. Movchan (215) noted that most fresh waters in the Ukraine are polluted and degraded by human activities. Habitat degradation is one of the main reasons for declining populations of native crayfish in the whole of Europe (Chucholl & Schrimpf, 215; Fűreder et al., 26). Two localities visited were considered unsuitable because the water temperature did not exceed 15 C and 17 C, respectively, in late summer and the degree days were probably not adequate for gonadal development and maturation of either crayfish species (Pursiainen & Erkamo, 1991). Narrow clawed crayfish were also found at several locations on the Crimean Peninsula (channel and ponds no. 31, 32, 35, 39 and 41; Table S3 Supporting Information) which were visibly affected by summer drying, by excessive deposition of silt and other sediments or by bank erosion caused by high flow rates in the spring and autumn, and by sewage inputs, agricultural pollution and soil erosion. These conditions must cause extreme and fluctuating water quality and environmental conditions over time. The presence of A. leptodactylus under these conditions supports the suggestion that it has wide ecological plasticity (Fűreder et al., 26; Holdich et al., 26; Kouba et al., 215; Kozák et al., 211; Pöckl et al., 26). In the Dnieper River, several Ukrainian national programmes have tried to improve catchment and habitat protection and eliminate pollution sources with the aim of improving its water quality and ecological conditions and eliminating environmental degradation and natural resources exploitation, in the context of protecting the Azov Sea and Black Sea stocks and environment (Movchan, 215). As well as habitat degradation, thick and narrow clawed crayfish in the Dnieper River are now threatened by the occurrence of nonnative marbled crayfish in a flooded quarry near Dnepropetrovsk city, which is close to the Dnieper River and its catchment (Novitsky & Son, 216). The pet trade and stocking of ornamental non native freshwater animals including marbled crayfish to garden ponds, currently poses a high risk of biological invasion and threat to the native aquatic biota (Patoka, Bláha, Kalous, & Kouba, 217). Illegal and unregulated exploitation of thick clawed crayfish by local fishermen without any fishing licence or permit presents the most serious threat to this species in Ukraine. A small proportion of thick clawed crayfish ( %) was found in total crayfish catches at all localities. Fishermen did not recognize thick clawed crayfish as a different crayfish species from the predominant narrow clawed crayfish in catches. An alarming discovery, confirmed in local fish markets, was that all thick clawed crayfish caught were intended for sale in the market and then for consumption. Unfortunately, the current Ukrainian fishery legislation and regulation is apparently of limited use in practice. Widespread unemployment and low living standards stimulate poaching and uncontrolled use of water bodies. This situation is exacerbated by a very limited Ukrainian state budget for wildlife and fishery management and fishery research, with only a few enthusiastic and professional staff employed in this sector (Movchan, 215). 4.7 Future needs for thick clawed crayfish conservation in Ukraine The rare and threatened status of A. pachypus in Ukraine emphasizes the need to define a clear and effective conservation strategy that will use all the principles of current conservation of European native crayfish species published by Fűreder et al. (26), Reynolds et al. (26) and Souty Grosset et al. (26). The most important requirements of thick clawed crayfish conservation for the future are (1) to change Ukrainian legislation with the aim of totally protecting thick clawed crayfish in all stages and decreasing the exploitation of aquatic biota (Movchan, 215), (2) to eliminate habitat degradation by organic and chemical pollution, alteration, fragmentation, intensive land use, agricultural and urban development (Chucholl & Schrimpf, 215; Fűreder et al., 26), (3) to avoid stocking of non native crayfish into Ukrainian waters and mitigate their adverse impact on native crayfish (Chucholl & Schrimpf, 215), (4) to improve and increase nature conservation actions in Ukraine, including educational and socio economic aspects (Movchan, 215), and (5) to apply basic and applied scientific approaches in understanding the biology, management and conservation of native crayfish species (Kozák, Füreder, Kouba, Reynolds, & Souty Grosset, 211). ACKNOWLEDGEMENTS The authors of this study heartily thank Professor Julian Reynolds (Ireland) for his scientific and English corrections and input. The study was supported financially by the Ministry of Education, Youth and Sports of the Czech Republic projects: CENAKVA (No. CZ.1.5/ 2.1./1.24), CENAKVA II (No. LO125 under the NPU I program) and GAJU 6/216/Z.

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