Effect of trophically different water bodies on the straminipilous fungal infection of Stenodus genus ( Coregoninae ) eggs

The authors investigated the growth of straminipilous organisms on the eggs of inconnu (Stenodus leucichthys) and nelma (Stenodus nelma) in water from three different eutrophication levels. Thirty (30) straminipilous species were found growing on the investigated eggs (of both fish species) used as baits. The majority of species has been found on the eggs of inconnu (20) in comparison with those found on the eggs of nelma (15). The highest number of infected eggs of both investigated species has been observed also in the water from Biała river (27.3% of the inconnu and 21.6% of the nelma; the most eutrophication), the smallest in water from Supraśl River (6.3 and 8.8% respectively; the less eutrophication). These differences for both species were statistically significant. Amino acid, carbohydrate and urease tests were used.


INTRODUCTION
Water eutrophication, river pollutants and shrinkage of natural reproduction places are the main causes of population drop of many fish species.Intensive fish harvesting also causes severe reductions in fish populations, especially of those species with a long sexual maturation period.Salmonid fishes, particularly the economically valuable species including the genus Stenodus (Letichevsky, 1983), belong to this group.Stenodus leucichthys, which is found in the Caspian Sea basin and spawns in rivers, notably the Wolga River, and Stenodus nelma (Kottelat and Freyhof, 2007;Bogutskaya and Coad, 2009;ITIS, 2010), which is found in the northern rivers of Eurasia (coming from Ponoja River and Onega Lake in the west to the Jukon and Mackenzie rivers in the east) belong to the genus Stenodus (Ladiges and Wogt, 1979;Page and Burr, 1991;Himberg and Lehtonen, 1995).Because of the impacts on fish populations, fish farms that use artificial reproduction are becoming more important.The predominant problem in all hatcheries is the infection of fish eggs by aquatic fungi (Mueller, 1994;Kitancharoen et al., 1997).Lartzeva and Altufiev (1987) and Czeczuga and Muszynska (1998) studied saprolegniasis on the eggs of S. leucichthys.
The resulting fungal infection of fish eggs of many freshwater species is a major economic problem for farmed fishes.Artificial reproduction of sturgeonid fishes has a high egg mortality rate due to saprolegnia fungus infection (Dudka et al., 1989), which can reach up to 70-90% of incubated eggs (Lartzeva and Altufiev, 1987).Hatai and Hoshiai (1992)  coho salmon (Oncorhynchus kisutch) and demonstrated heavy losses up to 50% during breeding of this species as a result of Saprolegnia parasitica.In the northwest USA, up to 22% of mature chinook and steelhead salmon returning to rivers died due to infected head wounds, which could perhaps explain the decline in salmon stocks to some extent (Neitzel et al., 2004).

investigated saprolegniosis of
In the present study, we investigated the fungus species that grow on the eggs of both Stenodus species and the effect of throphically different water bodies on the straminipilous fungal infection of Stenodus eggs.

MATERIALS AND METHODS
We used the eggs of S. leucichthys Guldenstädt (1772) (common name, inconnu) and S. nelma Pallas (1773) (common name, nelma).The eggs of inconnu were obtained from hatcheries of the Institute of Fisheries in Astrachan (Russia), while the eggs of nelma were collected from females from the upper stretch of the Angara River.Eggs were placed in a thermos flask with a physiological solution and were transported to our department by airmail.In the laboratory, the eggs (750 for each species) were washed three times in sterilized distilled water before being used for the experiments (Paterson and Bridge, 1994).

Study area
Two rivers and one pond located in the northeastern region of Poland (53° 07'N, 23°10'E-53°13'N, 23°20'E) were chosen for the study: 1. Biala River, length 9.8 km, width 3.7 m, depth 0.85 m, a left-bank tributary of the Suprasl River, which flows through the city of Bialystok.2. Suprasl River, length 93.1 km, width 6.0 m, depth 1.1 m, the largest right-bank tributary of the middle Narew River, which flows through the Knyszynska Forest 3. Komosa Pond, area 3.2 ha, maximum depth 1.25 m, surrounded by dense coniferous trees of the Knyszynska Forest.

Hydrochemical analyses
Water samples for the experiments were collected from these three different water bodies, and 19 hydrochemical variables were determined (Table 1) based on generally accepted methods (APHA, 2005).

Microbial and chemical analysis
The following procedure was used for determining the presence of straminipilous Peronosporomycetes (Oomycota) species on the Stenodus eggs (after fertilization).Water samples (800 ml each) from the different studied sites were placed in 1-L vessels, and 20-30 eggs were transferred to each vessel, as recommended by the general principles of culture (Watanabe, 2000).All vessels were enclosed in Petri scales with the bed turned upside down to prevent possible airborne contamination by mycotal spores.The vessels were stored at 15 ± 2°C with access to daylight to mimic natural conditions and following recommended guidelines (Seymour and Fuller, 1987).pH of the water has been analysed in every vessel.
All water analyses and experiments were carried out three times.Egg-covered mycelia (from each vessel) were observed every 3-4 days under a light microscope for 1 month, and the presence of morphological structures (sporangium, zoospores, antheridia, and oogonia) of straminipilous organisms was recorded.The taxa of the straminipilous species were identified using the keys of Johnson et al. (2005), Pystina (1998), andDick (2001).Saprolegnia parasitica was identified from the bundles of long hairs found on the secondary cysts and its indirect germination (Willeughby and Pickering, 1977).Tests for mino-acids, carbohydrates, and urease were carried out for the genera Achlya, Aphanomyces, Leptolegnia, Pythium and Saprolegnia, according to Yuasa and Hatai (1996) and Kitancharoen and Hatai (1998).For the carbohydrate utilization test, the medium used to culture the mycotal isolates was yeast nitrogen-based agar (Difco); GY (glucose-yeast extract) agar was used for the urease test.To prevent the growth of bacteria, ampicillin and streptomycin were applied to specific dilutions.The basal medium used in the amino acid assimilation test was the same as that used for the carbohydrate assimilation test.Bromo thymol blue and phenol red added to the yeast nitrogen-based broth and GY broth, respectively, were used as indicators.A positive result was determined by the change in colour of the medium to pink or purple; a change to orange and yellow was considered a negative result.These methods are described in detail in Czeczuga et al. (2011b).The results were tested for significance by analysis of variance and evaluated by the S-Scheffe test (Winer, 1997).

RESULTS
The hydrochemical variables of the water determined in the experiment are shown in Table 1.Water from the Biala River and Komosa Pond was the most eutrophic, whereas the water of the Suprasl River had the lowest content of all forms of nitrogen and phosphates.The highest levels of chemical oxygen demand (COD), CO 2 , chlorides, sulphates and calcium were found in the Biala River water.The water from Komosa Pond contained the highest levels of magnesium and iron.
Thirty (30) straminipilous organisms, including 23 belonging to the Saprolegniales, six to the Pythiales, and one to the Leptomitales, grew on the eggs of both species (Table 2).The genera Achlya, Saprolegnia and Pythium were the most common.The greatest number of species was found on the eggs of inconnu (20) as compared to species on nelma (15).The most commonly encountered species on the investigated eggs of both coregonid species were Saprolegnia parasitica, Saprolegnia ferax, Achlya polyandra and Pythium diclinum.Leptomitus lacteus, Pythium diclinum, and Pythium monospermum were rarely found.
The highest number of straminipilous species were found in the water from the Biala River (most eutrophic), while the lowest number were found growing on the eggs in containers with water from the Suprasl River (less eutrophic).The highest number of infected eggs of both species was found in water from the Biała River (27.3% of inconnu and 21.6% of nelma), while the lowest number was found in water from the Suprasl River (6.3 and 8.8%, respectively).
All species of Achlya, Aphanomyces, Leptolegnia and Saprolegnia genera assimilated glucose and starch (Table 3) but did not assimilate arabinose and salicin.Urease was only assimilated by species from the genera Leptolegnia, Pythium and Saprolegnia.Species of Achlya, Aphanomyces, Leptolegnia and Saprolegnia assimilated alanine but did not assimilate methionine, lysine, ornithine, leucine and glycine.
The total number of species and the number of infected eggs of both species for each water body were significantly higher at Biała River (the most eutrophication), the smallest in water from Supraśl River (the less eutrophication) (Table 2).

DISCUSSION
The genus Stenodus belongs to the family Salmonidae, with about 20 species in the subfamily Coregoninae (Bogutskaya and Coad, 2009;ITIS, 2010).They differ in size from relatively small, such as vendace (Coregonus albula L.), to the largest, nelma (Stenodus nelma), which can grow to 150 cm and 40 kg weight.Inconnu can grow to 110 cm and 20 kg weight (Rass, 1983;Svetovidov, 1984).Sexual maturation of both species is relatively late, nelma after 8-10 years and inconnu after 6-7 years from hatching.Nelma mostly lives in rivers, whereas inconnu is found in the Caspian Sea, but both species spawn in the upper course of rivers.
The highest number of straminipilous species was found to grow on the eggs of both species in the Biala River water, and the lowest numbers were found in the water from the Suprasl River; these differences were statistically significant.The Biala River is more eutrophic than the Suprasl River, and we have observed the same differences between rivers for straminipilous growing on the eggs of acipenserid fishes (Czeczuga et al., 2011c).
The environmental conditions of a water body (during the spawning period and during growth of salmon juveniles before migration to the sea) and the influence of stressogenic factors on both reproduction and fish growth (Iwama et al., 1997) are of great significance.Czeczuga and Muszyńska (1998) investigated the effect of environmental factors of 10 lakes in northeastern Poland on the growth of fungi, using the eggs of vendace (C.albula) and whitefish (Coregonus lavaretus L. generosus) collected in November.Results indicated that the eggs of vendace and whitefish from the various lakes were a habitat for 15-20 and 15-22 different fungi, respectively.Saprolegnia parasitica was found on vendace eggs from only three lakes, while Aphanomyces laevis and S. parasitica occurred on whitefish eggs collected from all 10 lakes.A comparison of data of eggs obtained from  vendace and whitefish in 1994 and 1995 indicated differences in the occurrence of fungi, possibly because of different environmental conditions between the two years that affected the condition of females during the prespawning period, egg quality, and/or the presence of spores of particular fungal species.Environmental stressors considerably attenuate fish immunity, as in the case of salmonid species (Maule et al., 1989).
Undoubtedly, the breeding of whitefish in tanks is a significant stressor that reduces their immunological status.For example, two species of trout, brook trout in their natural habitat were more resistant to Saprolegnia mycotic infections than pond-bred brown trout (Wald and Wizbach, 1992).Thus, the condition of females before spawning can affect the quality of eggs (Maule et al., 1989).Stress leads to a reduction in the quality of the eggs, which become smaller and less resistant to infections (Campbell et al., 1994).
The immunity of a given population is also food dependent (MacPhee et al., 1995).Sakai et al. (1992) found that chitin in trout food can stimulate fish resistance.The chitinous armour of crustaceans are known to be rich in carotenoids (Czeczuga et al., 2013), and interestingly, fewer fungi were found on the eggs of vendace, which contain more carotenoids, than on eggs from the same lake but with fewer carotenoids (Czeczuga and Muszyńska, 1998).This could explain the lesser number of fungi growing on the eggs of C. lavaretus, which contain more carotenoids (Czeczuga and Muszyńska, 1998).Carotenoids may serve as a source of oxygen in low-oxygen environments, ensuring a better state of the eggs and enhancing their resistance to mycotic infections (Karnaukhov, 1973).Salmonid species are oxygen dependent, and eutrophic waters contain less oxygen than water with a lower level of eutrophication.Thompson et al. (1994) showed that diet containing vitamin A increases the resistance of Atlantic salmon (Salmo salar L.) individuals.Carotenoids are known to be the precursors of vitamin A.
Studies have also investigated the significance of the genetic factor and resistance (Maule et al., 1989) of salmonid populations to both bacterial diseases (Beachman and Evelyn, 1992;Gjedrem and Gjöen, 1995) and mycotic infections (Nilsson, 1992).Results of the investigations on the assimilation of amino acids, carbohydrates and urease by fungi are similar to records for other salmonid fishes (Kitancharoen and Hatai, 1998;Czeczuga et al., 2011b) and for species from other families (Yuasa and Hatai, 1996).Straminipilous from the order Saprolegniales, especially Saprolegnia, Achlya, Aphanomyces and Dictyuchus species, are responsible for the egg infection in investigated Stenodus species.Amongst the Saprolegniales, Aphanomyces frigidophilus, Dictyuchus pisci, Saprolegnia polymorpha and Saprolegnia salmonis are rare species to fishes. A. frigidophilus and S. salmonis were first described in Japan in salmonid fishes (Kitancharoen and Hatai, 1997;Hussein and Hatai, 1999), and D. pisci was described from India (Khulbe, 1994).S. polymorpha was first described by Willoughby (1998) in ornamental carp (Cyprinus carpio L. koi) in the United Kingdom.All four species occur in water bodies of Poland.A. frigidophilus and S. salmonis have been found on the eggs of C. lavaretus, Salmo trutta L. (Czeczuga et al., 2005a), and Salmo salar (Czeczuga et al., 2011a) and also on the eggs of some other sturgeon species (Czeczuga et al., 2011c).Dictyuchus pisci and S. polymorpha have also been found on the eggs of some sturgeonid species (Czeczuga et al., 2011c) and S. polymorpha in the muscles of Carassius auratus (L.) adults (Czeczuga et al., 2011d).
phytes (Czeczuga et al., 2005b).S. parasitica was one of the most frequently occurring species on the examined eggs of both Stenodus species.
The lifecycle of S. parasitica consists of a sexual and asexual phase (Figure 1).Hyphal cells form the sexual structures of antheridia and oogonia, which release motile oospores.After germination, these oospores release a new mycelium.These sexual structures have been found in taxonomical investigations of several species of Saprolegniales (Dick, 2001).The asexual sporangia and spores are formed at the end of hyphae, and this releases many motile zoospores (Bruno and Wood, 1999), which are the primary zoospores.These swim for a short time and encyst in secondary zoospores, which are motile for a longer period and have been considered to be the main infectious spores (van West, 2006).After encystment, the cyst releases a new secondary zoospore.The secondary cyst of Saprolegnia species can possess hairs, which in S. parasitica contain the "boat hooks."The connection between the secondary zoospore and secondary cyst forms a new cyst, a process called polyplanetism (Beakes, 1983).Secondary cysts are an infective form for fishes.
Investigations have been carried out on the environmental factors influencing the zoospores from different Saprolegnia species (Smith et al., 1989), the morphological features of the vegetative hyphae (Ali, 2005), and the ultrastructure of Saprolegnia secondary zoospore cyst ornamentation (from infected wild brown trout, indicating two distinct morphotypes amongst longspined species) (Fregeneda-Grandes, 2000).The morphology of the cyst of the first morphotype shows a higher number of bundles with a greater number and length of hair, whereas the cysts from the second group have fewer bundles and less and shorter hair.Cysts of the saprolegniosis isolated from the salmonids in our study belonged to the second group, whereas most isolates from fish muscle and river water belonged to the first group (Fregeneda-Grandes, 2000).In the genera Saprolegnia and Isoachlya, hair has been found on the cyst of secondary zoospores (Pickering et al., 1979;Beakes, 1983;Hallet and Dick, 1986).Other studies proved that the isolates pathogenic to salmonids have long hair on the secondary cysts, terminating in hooks and grouped in bundles; in contrast, the saprophytes have much shorter hair, which are not grouped in bundles (Pickering et al., 1979).Transmission electron microscopy of Saprolegnia secondary cysts showed variations in the hair termination (Fregeneda-Grandes, 2000): hair without hooks, hair with one to three hooks, and hair with two asymmetrical hooks.
The presence of the secondary cysts with hooked hairs has been considered an adaptation for attaching to a substrate, especially to a fish surface, their eggs, and other aquatic organisms (Meyer, 1991;Pickering and Willoughby, 1982).Studies on the morphology of the secondary cysts of pathogenic Saprolegnia species from different geographical origins (Fregeneda-Grandes, 2000;Stueland et al, 2005) have shown that the number of bundles per cyst, the number of hair per bundle, and hair length are related to different environmental conditions and to the surface of the aquatic host.
Chemical characterization of the zoospores and cysts of saprophytic and fish-pathogenic Saprolegnia species also differs (Burr and Beakes, 1994).Water salinity significantly reduced the total free amino acids and protein content of mycelia of the fish pathogen S. parasitica (Ali, 2005).In addition, ITS rDNA sequences of the Saprolegnia diclina-S.parasitica complex from diverse geographical origins have shown physiological and morphological differences and variations in their pathogenicity (Dieguez-Uribeondo et al., 2007).Adaptation to parasitism in Saprolegnia species might also have occurred at the spore level by the development of long-hooked hairs to facilitate host attachment.Differences in the number of morphological and physiological straminipilous forms and in the phylogeny of the Saprolegnia species based on ITS rDNA sequences have been observed.Some parasites translocate proteins to its host cells, thereby modifying the hosts' molecular processes.This phenomenon has been found in the malaria (Plasmodium) parasite (Romisch, 2005), which translocates proteins to red blood cells.Saprolegnia species and other oomycetes may have evolved a similar protein translocation system (Birch et al., 2006).According to van West (2006), S. parasitica species have two translocating proteins, which might be responsible for suppressing the immune response of their host.

Table 1 .
Chemical and physical properties of water in particular water bodies (in mg l -1 ).

Table 2 .
Number of straminipilous organisms recorded on the eggs of Stenodus species in water of particular water bodies.
cMeans with the same letter are not significantly different (p>0.05).

Table 3 .
Amino acid, carbohydrate and urease assimilation by straminipilous organisms isolated from eggs of Stenodus species.