Histopathology of the Black Plaice Pseudopleuronectes Obscurus From a Marine Reference Area

The Kievka Bay is one of the cleanest regions on the Far East coast of Russia. There are no anthropogenic impacts on the coastal area, and thus the infl uence of pollutants on the state of fi sh health is minimized. Histopathological examination of black plaice was performed to study the background level of morbidity and the occurrence of pathological alterations in tissues of fi shes from this ecologically clean region. Different pathological alterations were detected in fi shes sampled. The most surprising was a disease of black plaice, where necrotic changes were found in the gonads of 28% of the males, and the destructive changes were absent in females. In the gills, pathogenic microorganisms were detected, which induced strong hyperplasia of the respiratory epithelium, fusion of lamellas, and necrosis of pillar cells, in 28% of the individuals. Diseases of the liver were recorded for two fi shes that comprised 8% of the total number, in one case – granulomatous infl ammation. Besides that some fi shes had a diffuse necrosis in the liver. In the kidneys of all sampled fi sh, parasites were present in the lumen of the renal duct, very likely a moderately pathogenic species, as the consequences of parasitism were insignifi cant and for a smaller part of fi shes – moderate. Thus, different pathological alterations were found in all investigated organs, and, in most cases, it was possible to establish their occurrence with parasitism. Research Article


Introduction
Flatfi shes are widely used as bioindicators of contamination of the marine environment [1][2][3][4] including moderate and heavily contaminated parts of Peter the Great Bay, Sea of Japan [5]. In our previous investigations we used endemic speciesbarfi n plaice Liopsetta pinnifasciata and one of the most common species -black plaice Pseudopleuronectes obscurus [6]. The black plaice is a typical inhabitant of the upper subtidal. P. obscurus spends the greater part of its life in the near-shore zone, where various pollutants concentrate. Hence, it serves as an indicator species for the quality of the aquatic environment in Peter the Great Bay [7]. In terms of many parameters, the black plaice is more sensitive to pollution of marine environment than other species. Nevertheless, the occurrence of histopathological changes in this species from uncontaminated waters has not been studied. The aim of the present research was to evaluate, using biological, morphological, and histopathological indices, the state of the internal organs and tissues of fi sh from an uncontaminated reference marine area in Kievka Bay, Sea of Japan, in a case study of the plaice P. obscurus.

Materials and Methods
The investigation was conducted in a marine reference area Total length (TL), standard length (SL) and total body weight (W) were recorded for each animal. During the dissection the ovaries and liver were removed and weighed.
Gonadosomatic index (GSI) was expressed as the percentage of body weight represented by the gonads: GSI (%) = (gonad weight/total weight) × 100. All values are expressed as the mean ± standard deviation (M ± SD). Data on CF, HSI and GSI are presented for fi sh collected in 2006, no signifi cant changes of these parameters were found in 2007, so data are not shown.
Tissue specimens of liver, kidney, gills and gonad (0.8 × 0.8 см) were fi xed with Bouin's fl uid for 48 h, dehydrated through a graded series of increasing concentrations of ethanol, cleared in xylene and embedded in paraffi n. Sections (5-6 μm thick) were stained with eosin-hematoxilin by the standard method.
The state of the tissues and organs were examined under a light microscope Olympus BX41 (Olympus, Japan) equipped with digital camera C5060-ADU (Olympus, Japan). Occurrence and classifi cation of parasites in histological slides was established according to recommendations [8]. Liver tissues were analyzed for the presence of parasites, degenerative, infl ammatory, necrotic, and preneoplastic changes, which were identifi ed by generally accepted criteria [2,9].

Results
Condition factor of black plaice caught from 25 June to 1 July varied from 1.97 to 3.38, with a mean value of 2.44 ( Figure   1). HSI ranged from 0.59 to 2.96, the mean in females 1.74 ± 0.66 (N = 9), and in males 1.48 ± 0.43 (N = 17). GSI ranged from 0.05 to 2.28. Low values of GSI are characteristic for fi sh in the "rest stage", higher GSI up to 2.28 was found in two females in the "beginning of vitellogenesis stage". In females, the mean GSI was 1.05 ± 0.61 (N = 9), and in males, it was 0.20  15 13 12 23 16 14 3 9 27 17 20 25 11 26 28 4 21 6 19 8 18 2 7 1 24 22 5   with the prevalence about identical at 28%. On the diseases of fi sh from this water area is not known, but there is evidence that in the sea urchin Strongylocentrotus intermedius found spawning failure [11].
The pathology of the gonads has been frequently recorded in fi sh inhabiting the contaminated areas. Cases of large increase or decrease in gonad volume (sometimes their full absence), asymmetry of the paired gonads (at worst -the development only of one), and asynchronous maturation have been described. In male gonads, connective tissue constrictions can be formed and, as a consequence, -the formation of the twisted gonads, tucks, and segmentation. In some cases, dense  , their volume increase (hypertrophy) was also marked. The nuclei of Sertoli cells in abnormal testis were augmented by 25% as contrasted to those in a normal gonad. Spermatogenesis was lacking with only single spermatogonia detected. This type of testis pathology very seldom occurs in fi sh. Isolated instances are described and identifi ed by the terms hypogonadism [15] and testis dysgenesis [16]. Thes e types of testis pathology are not characteristic for P. obscurus from Kievka Bay. Thus, the testis disease in black plaice from Kievka Bay was detected for the fi rst time. The necrotic changes were observed only in the gonads of males, not females. Comparative analysis of histopathological observations of the testis of black plaice from Kievka Bay shows that a similar disease was not seen in black plaice or in other fl atfi sh species from Peter the Great Bay.
Pathology of the gonad of fi sh from contaminated areas is characterized by specifi c types of changes, in particular in phenotypic male fl ounder Platichthys fl esus from the Mersey estuary (UK) impacted by endocrine disrupting chemicals, uniand bilateral intersex gonads with abnormal ovarian follicular components in the testis were observed [17]. Reduced testicular growth and sperm production was observed in male fi sh under experimental exposure to contaminants, for example in male rainbow trout Oncorhynchus mykiss exposed to four different alkylphenolic chemicals [18]. Very specifi c pathology of the testes or sperm ducts was observed in spotted gar Lepisosteus oculatus from Bayou Trepagnier, Louisiana, USA, a petroleumcontaminated site, multilocular fl uid-fi lled cysts [19].   [20] and Henneguya testicularis [21]. The fi rst of these parasites, S. testicularis, induces the destruction of seminiferous tubules, the decrease in a number or full elimination of mature spermatozoa.
The pathogenic effect of the second species, H. testicularis, was not indicated. Additional studies are required for detection of a possible pathogenic agent of the testis necrosis noted in this study.
There were granulomas in the kidneys and, in one case, the liver of black plaice from Kievka Bay. The formation of multiple granulomas is denoted by the term a granulomatosis.
Granulomatosis is a nonspecifi c type of an infl ammation that is induced in fi shes by a variety of the different agents. A classic example is the infl ammatory response in fi shes, as well as in more highly organized vertebral animals, to bacterial infections by the genus Mycobacterium [22,23]. Granulomatosis is induced also by other organisms, in particular some endocellular bacteria, Dermocystidium-like parasites [24] and Ichthyophonus hoferi [25]. The formation of a granuloma contributes to the control of pathogens, which cannot be eliminated by phagocytosis or digested by macrophages. The granulomas in fi sh represent the compact, organized accumulation of mature monocytic phagocytes (macrophages and/or epithelioidal cells), although the cellular composition of granulomas can vary. In a black plaice, the forming of granulomas was tracked by a powerful infi ltration of enclosing tissues by leucocytes that suggests a strong immune response. Almost all detected granulomas had a necrotic center.

Conclusion
Different pathological alterations were found in organs of black plaice from Kievka Bay, northwestern Sea of Japan. In most cases it was possible to associate these alterations with parasitism. The principal fi ndings are necrosis in the testis, hyperplasia of the respiratory epithelium, fusion of gill lamellas, necrosis of pillar cells in gills, and granulomatous infl ammation in the kidney and liver. The spectrum and occurrence of histopathological changes were lower than in fl atfi sh from contaminated parts of Peter the Great Bay, Sea of Japan. Several of the observed abnormalities in this study were found in fl atfi sh from Russian part of the Sea of Japan for the fi rst time.