WSN 95 (2018) - World Scientific News

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WSN 95 (2018) 21-51

EISSN 2392-2192

Estimation of physiological and biological indices and consequences of biological invasion of the pumpkinseed Lepomis gibbosus (Linnaeus, 1758) in the Zaporizke Reservoir, Ukraine Marenkov Oleh*, Nesterenko Oleh Department of General Biology and Water Bioresources, Oles Honchar Dnipro National University, P.M.B. 49050, Dnipro, Ukraine

*E-mail address: [email protected]

ABSTRACT Due to the emergence of new species in ichthyofauna of the Zaporizke reservoir, there was a need to study their biology, ecology and patterns of their distribution. Biological invasions of new species of fish can have diverse effects both within aboriginal populations and within the entire ecosystem. The pumpkinseed Lepomis gibbosus (Linnaeus, 1758) is one of the most common alien species in the Zaporizke reservoir, which has already adapted to the new environment and quickly develops the reservoirs of the region and increases its number. In addition, this species has a wide range of nutrients and can compete for feed resources with other species of fish. The purpose of the work: to study the biological and ecological parameters of the pumpkinseed, the basic laws of its physiology. The paper describes the results of fish morphological studies, estimation of the number and biomass of young fish, determines the morphometric parameters of peripheral blood, determines fertility indexes and features of the course of oogenesis. The paper presents preliminary results of the assessment of the consequences of the invasion of this species in the basin of the Zaporizke Reservoir. Keywords: pumpkinseed, Lepomis gibbosus, Zaporizke Reservoir, Dnipro, erythrocytes, fish egg, biological invasion

( Received 08 February 2018; Accepted 24 February 2018; Date of Publication 25 February 2018 )

World Scientific News 95 (2018) 21-51

1. INTRODUCTION Due to the appearance of new species in ichthyofauna of the Zaporizke Reservoir it is necessary to study their biology, ecology and distribution patterns. Biological invasions of new species of fish can have diverse effects both within indigenous populations and within the entire ecosystem. The pumpkinseed Lepomis gibbosus (Linnaeus, 1758) is a member of the sunfish family (Centrarchidae) of order Perciformes [1, 2]. The natural range of the pumpkinseed is the freshwater reservoirs of North America from the Great Lakes to Florida. This fish prefers quiet reservoirs with sandy or pebble soil. At the end of the XIX century a pumpkinseed was introduced to Western Europe as an aquarium fish. From the ponds in which it was grown, L. gibbosus (Linnaeus, 1758) has reached the basins of small, and later large rivers of Europe and Asia [3, 4]. Currently, the pumpkinseed is one of the most common species of alien species in the Zaporizke Reservoir [5]. Given that this species has already adapted well to the new environment, it quickly adapts in the reservoirs of the region and rapidly increases its number [6, 7]. Also, this species has a wide range of nutrition and can compete other species of fish for feed resources. Therefore, the study of biology and the effects of invasion of this species is very relevant. The purpose of our work was to study the biological and ecological parameters of the pumpkinseed, the basic regularities of its physiology.

2. MATERIALS AND METHODS Researches were conducted in The Zaporizke (Dniprovske, Dnipro, Dnieper) Reservoir – the multi-purpose reservoir during 2013–2017. The reservoir is located in the South-west of Ukraine, in the territory of the agro-industrial zones and been under strong anthropogenic influence. 2. 1. Characteristics of the research area Zaporizke Reservoir belongs to the channel type. Type of reservoir by genesis and location is plain-river; by configuration it is channel; by volume and area it is large; by depth it is mid-depth; by water exchange it has a very large exchange [8]. Zaporizke Reservoir is located on the territory of Dnipropetrovsk and Zaporizhia regions of Ukraine. The water reservoir was created on the rapid and middle sections of the Dnieper in 1931–1934 as a result of the construction of the Dnieper Hydroelectric Station (DniproHES). The reservoir is limited by dams: below by DniproHES in the city of Zaporizhia (47°52′09″N 35°05′13″E), and in the top by The Middle Dnieper Hydroelectric Station in the city of Kamianske (48°32′52.80″N 34°32′28.67″E) [8, 9]. Right bank of the reservoir is located in the steppe area of Pridneprovsk high ground. The left bank of the reservoir is located in the steppe region of the Pridneprovsk lowland, divided into two subregions: the northern one, characterized by a combination of valley flooded areas, meadow and swamp forest steppe types with weak dismemberment (the Dnipro valley) and southern, extending from the mouth of the Samara River to the dam of DniproHES and is characterized by the broad development of girder-beam types of terrain [8].

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The length of the reservoir is 128.5 km, the total length of the coastline is about 250 km. The maximum depth at the dam of the DniproHES is 60 m. The regime of regulation occurs dayly and weekly, level fluctuations are up to 0.7 m [8, 9]. The physico-geographical zoning of the Zaporizke Reservoir is based on the general principles of zoning of reservoirs using existing analogies with the zoning of some Volga and Dnipro reservoirs. The factual basis of zoning was the maps of the area of the reservoir bed until it was flooded, pilotage water reservoir maps, hydrometeorological yearbooks and information on geomorphology and hydrology [8, 9].

Figure 1. Physico-geographical position of the Zaporizke Reservoir (48°08′09″N 35°07′43″E).

Due to the fact that the reservoir is formed mainly in flooded valleys of the Dnipro and Samara (Fig. 1), its water area is divided into 2 ples: Main Dnieper and the Samara Bay (stretching for approximately ten kilometres at the mouth of the Samara River, flows into the -23-


northern end of the reservoir). For the first time, the water distribution of Zaporizke Reservoir began T.O. Svirenko [8]. Considering the structure of the Dnieper valley, the morphology of the bed and the degree of flooding, it divided the reservoir into 2 stretches: the upper one is from the place of lens out, of the affluent (Verkhnedneprovsk) to the former rapidshare; the lower one is the former cataracted area. Based on the speed of the flow and hydrobiological indicators, the Zaporizke Reservoir is divided into three sections: the upper one is river; middle one is transitional; the lower one is lake [8]. Thus, these divisions give both geographic and hydrobiological basis for the research. The reservoir is a multipurpose object with daily and weekly regulation and pre-loading operation. During the annual level, there are periods of spring-summer filling, summerautumn stabilization, winter weathering. 2. 2. Method of morphometric study Currently, the methods of complete morphometry in biological research are sufficiently effective and informative. The study of morphometric indicators of fish makes it possible to show how environmental factors can affect the structure and adaptation of fish. Differences in morphometric indices of even-aged, sexually mature individuals caught in different reservoirs can indicate an ecological factor that needs further in-depth studies [5, 10]. Biological analysis of fish was carried out in accordance with generally accepted ichthyological methods. Morphometric parameters were measured on a fresh material by such scheme (Fig. 2) [5, 10]. Morphometric analysis was performed on 22 plastic and 6 meristic features.

Figure 2. Scheme of the measure of the pumpkinseed [5].

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World Scientific News 95 (2018) 21-51 Following indicators were measured in fish: L – absolute length of fish; am –the length by Smith; l – industrial or ichthyological length; lcor – length of the body; lr – the length of the snout; do – the diameter of the eye; po – distance behind the eye; ls – length of the head; tt1 – head height near the neck; H – the highest body height; h – the smallest height of the body or the height of the tail stem; aD – antedorsal distance; pD – postdorsal distance; pl – the length of the tail stem; lD – base of dorsal fin; hD – the highest height of the dorsal fin; lA – length of the base of the anal fin A; hD – the highest height of the anal fin A; LP – length of pectoral fin P; lV – length of the ventral fin V; PV – pectroventral distance; VA – ventroanal distance; l.l – number of scales in the lateral line. D – number of rays in the dorsal fin; A – number of rays in an anal fin; P – number of rays in the ventral fin; V – number of rays in the ventral fin; C – number of rays in the tail fin. 2. 3. Morphometric analysis of blood smears The morphometric analysis of dyed smears of blood using a direct light microscope is still a key research in assessing the state of hematopoiesis. Many changes that arise as a result of the influence of external environmental factors on the organism of fish can be traced by changes in the morphological parameters of the blood formed elements. The distribution of erythrocytes by size of their diameter (anisocytosis) on microcytes, normocytes and macrocytes was proposed by Hayem G. in 1889. Further, the most widely used indicators of red blood cells were proposed by the Price Jones and they are reflected on the histogram, named after him, which shows distribution of red blood cells by diameter. Technically, Prices Jones has measured cells by projecting the microscope field of view with a blood on the screen, measuring their projection by a ruler in two diameters at a constant magnification scale. Other researchers have measured erythrocytes under microscope by Leitsevsky ocular micrometer. In addition, some authors favored the use of wet preparations, believing that erythrocytes thicken on fixed drugs. Other authors believe that fixation and coloring do not change the size of erythrocytes [11]. Thus, due to the popularity of the interpretation of the measurement of morphometric indices of erythrocytes, a single approach on this issue has not been developed. Computer analysis of images, which in recent years has become the most used, can be considered as a universal method for measuring biological objects, which allows obtaining data that is not available when using current hematologic analyzers. The morphometric parameters include: diameter (D), thickness (T), area (S), index of sphericity (Lsf) [11]. Investigation of the parameters of the formed blood elements of L. gibbosus (Linnaeus, 1758), namely erythrocytes, was carried out with several parameters: the area of the erythrocyte, the area of the nucleus, the small and large diameters of the red blood cell. Those parameters were measured in several age groups of L. gibbosus (Linnaeus, 1758): firs summer fish (0+), yearlings (1+), two years old (2+). The morphometric study of erythrocytes was performed using a computer analysis of the microphotographs made using the "Sciencelab T500 5.17 M" digital camera, which was attached to the microscope “Biolam 70”. 2. 4. Method of eggs research Fish eggs studies are sufficiently informative to determine the fertility and reproductive capacity in general. The main methods for eggs studies are the determination of the stage of maturity of the gonads and the fertility of the fish, as well as the histological study of the

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gonads. Study of data on gametogenesis is essential for a better understanding of the adaptive capacity of fish to conditions of existence and provide an opportunity to predict the population size, understand the laws of physiology of a certain species of fish. 2. 5. Method of determining fertility When sampling for fertility, each female was measured and weighed, and the scales were taken for further age determination. The fish were then dissected, the entire ovary was weighed and sample was separated from the for counting. The sample was weighed on pharmacist scales, placed in a container, labeled and filled with a weak 4% formalin. The species and serial number of fish, time and place, fishing gear, degree of maturity, body length, weight of all fish, spawn and selected sample were recorded to the corresponding biological journal. The graphs were left to enter the number of eggs in the sample, in the whole ovary, the diameter of the eggs and for the age of the experimental fish. To determine the average size of the egg, 10 eggs were taken, placed in a straight line and the length of this line was divided by 10, obtaining the average diameter of the eggs. As the eggs swells in water, eggs should be freshly removed from the ovary or fixed in the formalin. For each individual, the index of individual absolute fertility was calculated. Individual Absolute Fertility (IAF) is the number of mature eggs in the ovaries of one fish. Calculations were carried out using the weighting method using formula (1): IAF =g×∙n,

(1)

where: g – is the weight of ovary, g; n – is the number of eggs in 1 g of sample. 2. 6. Methodology for the study of oogenesis Ichthyological material was taken from end of spring till the beginning of summer, at a water temperature of 15 to 25 °C when the spawning of the pumpkinseed continues. Sections were made using microtome «MZP-01 Tehnom». Specimens were dyed by hematoxylin-eosin and by Mallory staining [12, 13]. Microphotographs of histological specimens were taken by digital camera «Sciencelab T500 5.17M», which was connected to the microscope “Biolam 70”. The oocytes were descripted as per M. M. Shihshabekov [14] (Fig. 3). Three phases of development of oocytes were chosen for the study: D, E and E–F: phase D is the phase, when the sex cells pass a stage of primary accumulation of yolk and vacuolation, a period of trophoplasmic growth; phase E is oocyte filled with yolk; phase E–F is when oocyte is in the transitional stage of development, before entering the environment [14]. Using special camera software, morphometric parameters of the oocytes were measured, namely: cell area, nucleus area, diameter and vacuole area, and the thickness of the oocyte membrane. During the work, bioethical norms are not violated. The experiment was performed according to the “Provisions for the use of animals in biomedical experiments” [15].

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Figure 3. Scheme of gametogenesis by M. M. Shihshabekov [14].

2. 7. Statistical processing Statistical data processing was carried out by conventional methods using software packages for personal computers Statistica 8.0 (StatSoftInc, USA). All results are given as the mean ± standard deviation (SD). There liability of the difference between data samples was determined using one-factor ANOVA dispersion analysis at a significance level p