Vol. 8(6), pp. 503-510, 5 February, 2014 DOI: 10.5897/AJMR2013.5963 ISSN 1996-0808 ©2014 Academic Journals African Journal of Microbiology Research http://www.academicjournals.org/AJMR

Full Length Research Paper

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

Bazyli Czeczuga*, Adrianna Semeniuk and Ewa Czeczuga-Semeniuk

Departament of General Biology, Medical University, Mickiewicza 2c, 15-222 Białystok, Poland.

Accepted 23 January, 2014

The authors investigated the growth of straminipilous organisms on the eggs of inconnu () 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.

Key words: Inconnu, Stenodus leucichthys, nelma, Stenodus nelma, fish eggs, straminipilous organisms, eutrophication.

INTRODUCTION

Water eutrophication, river pollutants and shrinkage of and Wogt, 1979; Page and Burr, 1991; Himberg and natural reproduction places are the main causes of Lehtonen, 1995). Because of the impacts on fish population drop of many fish species. Intensive fish populations, fish farms that use artificial reproduction are harvesting also causes severe reductions in fish becoming more important. The predominant problem in populations, especially of those species with a long all hatcheries is the infection of fish eggs by aquatic fungi sexual maturation period. Salmonid fishes, particularly (Mueller, 1994; Kitancharoen et al., 1997). Lartzeva and the economically valuable species including the genus Altufiev (1987) and Czeczuga and Muszynska (1998) Stenodus (Letichevsky, 1983), belong to this group. studied saprolegniasis on the eggs of S. leucichthys. Stenodus leucichthys, which is found in the The resulting fungal infection of fish eggs of many basin and spawns in rivers, notably the Wolga River, and freshwater species is a major economic problem for Stenodus nelma (Kottelat and Freyhof, 2007; Bogutskaya farmed fishes. Artificial reproduction of sturgeonid fishes and Coad, 2009; ITIS, 2010), which is found in the has a high egg mortality rate due to saprolegnia fungus northern rivers of Eurasia (coming from Ponoja River and infection (Dudka et al., 1989), which can reach up to 70- Onega Lake in the west to the Jukon and Mackenzie 90% of incubated eggs (Lartzeva and Altufiev, 1987). rivers in the east) belong to the genus Stenodus (Ladiges Hatai and Hoshiai (1992) investigated saprolegniosis of

*Corresponding author. E-mail: [email protected].

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

River Pond Specification River Supraśl Biała Komosa Temperature (°C) 17.8 17.0 17.4 pH 7.10 7.82 7.60 DO 6.4 11.2 12.8

BOD5 7.2 2.8 7.4 Oxidability (COD ) 15.82 6.60 13.20

CO2 26.90 6.60 8.30 -1 Alkalinity in CaCO3 (mval l ) 4.3 4.5 3.9

N-NH3 0.621 0.142 0.161

N-NO2 0.132 0.006 0.009

N-NO3 0.473 0.014 0.034

P-PO4 1.824 0.158 0.255

Sulphates (SO4) 73.24 32.38 42.75 Chlorides (Cl) 66.44 17.12 23.51 Total hardness in Ca 98.26 73.42 68.40 Total hardness in Mg 17.42 11.58 28.81 Fe 0.92 0.48 1.54 Dry residue 434.0 197.0 375.0 Dissolved solids 324.0 179.0 312.0 Suspended solids 110.0 18.0 63.0

coho salmon (Oncorhynchus kisutch) and demonstrated Poland (53° 07’N, 23°10’E-53°13’N, 23°20’E) were chosen for the heavy losses up to 50% during breeding of this species study: as a result of Saprolegnia parasitica. In the northwest 1. Biala River, length 9.8 km, width 3.7 m, depth 0.85 m, a left-bank USA, up to 22% of mature chinook and steelhead salmon tributary of the Suprasl River, which flows through the city of returning to rivers died due to infected head wounds, Bialystok. which could perhaps explain the decline in salmon stocks 2. Suprasl River, length 93.1 km, width 6.0 m, depth 1.1 m, the to some extent (Neitzel et al., 2004). largest right-bank tributary of the middle Narew River, which flows In the present study, we investigated the fungus through the Knyszynska Forest species that grow on the eggs of both Stenodus species 3. Komosa Pond, area 3.2 ha, maximum depth 1.25 m, surrounded by dense coniferous trees of the Knyszynska Forest. and the effect of throphically different water bodies on the straminipilous fungal infection of Stenodus eggs. Hydrochemical analyses

MATERIALS AND METHODS Water samples for the experiments were collected from these three different water bodies, and 19 hydrochemical variables were We used the eggs of S. leucichthys Guldenstädt (1772) (common determined (Table 1) based on generally accepted methods name, inconnu) and S. nelma Pallas (1773) (common name, (APHA, 2005). 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 Microbial and chemical analysis River. Eggs were placed in a thermos flask with a physiological solution and were transported to our department by airmail. In the The following procedure was used for determining the presence of laboratory, the eggs (750 for each species) were washed three straminipilous Peronosporomycetes (Oomycota) species on the times in sterilized distilled water before being used for the Stenodus eggs (after fertilization). Water samples (800 ml each) experiments (Paterson and Bridge, 1994). 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 Study area enclosed in Petri scales with the bed turned upside down to prevent possible airborne contamination by mycotal spores. The vessels Two rivers and one pond located in the northeastern region of were stored at 15 ± 2°C with access to daylight to mimic natural

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conditions and following recommended guidelines (Seymour and of inconnu and 21.6% of nelma), while the lowest number Fuller, 1987). pH of the water has been analysed in every vessel. was found in water from the Suprasl River (6.3 and 8.8%, All water analyses and experiments were carried out three times. respectively). Egg-covered mycelia (from each vessel) were observed every 3-4 days under a light microscope for 1 month, and the presence of All species of Achlya, Aphanomyces, Leptolegnia and morphological structures (sporangium, zoospores, antheridia, and Saprolegnia genera assimilated glucose and starch oogonia) of straminipilous organisms was recorded. The taxa of the (Table 3) but did not assimilate arabinose and salicin. straminipilous species were identified using the keys of Johnson et Urease was only assimilated by species from the genera al. (2005), Pystina (1998), and Dick (2001). Saprolegnia parasitica Leptolegnia, Pythium and Saprolegnia. Species of was identified from the bundles of long hairs found on the Achlya, Aphanomyces, Leptolegnia and Saprolegnia secondary cysts and its indirect germination (Willeughby and Pickering, 1977). Tests for mino-acids, carbohydrates, and urease assimilated alanine but did not assimilate methionine, were carried out for the genera Achlya, Aphanomyces, Leptolegnia, lysine, ornithine, leucine and glycine. Pythium and Saprolegnia, according to Yuasa and Hatai (1996) and The total number of species and the number of infected Kitancharoen and Hatai (1998). For the carbohydrate utilization eggs of both species for each water body were test, the medium used to culture the mycotal isolates was yeast significantly higher at Biała River (the most nitrogen-based agar (Difco); GY (glucose-yeast extract) agar was used for the urease test. To prevent the growth of bacteria, eutrophication), the smallest in water from Supraśl River ampicillin and streptomycin were applied to specific dilutions. The (the less eutrophication) (Table 2). 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 DISCUSSION broth and GY broth, respectively, were used as indicators. A positive result was determined by the change in colour of the The genus Stenodus belongs to the family , medium to pink or purple; a change to orange and yellow was with about 20 species in the subfamily Coregoninae considered a negative result. These methods are described in detail in Czeczuga et al. (2011b). The results were tested for significance (Bogutskaya and Coad, 2009; ITIS, 2010). They differ in by analysis of variance and evaluated by the S-Scheffe test (Winer, size from relatively small, such as vendace ( 1997). 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, RESULTS 1984). Sexual maturation of both species is relatively late, nelma after 8-10 years and inconnu after 6-7 years The hydrochemical variables of the water determined in from hatching. Nelma mostly lives in rivers, whereas the experiment are shown in Table 1. Water from the inconnu is found in the Caspian Sea, but both species Biala River and Komosa Pond was the most eutrophic, spawn in the upper course of rivers. whereas the water of the Suprasl River had the lowest The highest number of straminipilous species was content of all forms of nitrogen and phosphates. The found to grow on the eggs of both species in the Biala highest levels of chemical oxygen demand (COD), CO2, River water, and the lowest numbers were found in the chlorides, sulphates and calcium were found in the Biala water from the Suprasl River; these differences were River water. The water from Komosa Pond contained the statistically significant. The Biala River is more eutrophic highest levels of magnesium and iron. than the Suprasl River, and we have observed the same Thirty (30) straminipilous organisms, including 23 differences between rivers for straminipilous growing on belonging to the Saprolegniales, six to the Pythiales, and the eggs of acipenserid fishes (Czeczuga et al., 2011c). one to the Leptomitales, grew on the eggs of both The environmental conditions of a water body (during species (Table 2). The genera Achlya, Saprolegnia and the spawning period and during growth of salmon Pythium were the most common. The greatest number of juveniles before migration to the sea) and the influence of species was found on the eggs of inconnu (20) as stressogenic factors on both reproduction and fish growth compared to species on nelma (15). The most commonly (Iwama et al., 1997) are of great significance. Czeczuga encountered species on the investigated eggs of both and Muszyńska (1998) investigated the effect of coregonid species were Saprolegnia parasitica, environmental factors of 10 lakes in northeastern Poland Saprolegnia ferax, Achlya polyandra and Pythium on the growth of fungi, using the eggs of vendace (C. diclinum. Leptomitus lacteus, Pythium diclinum, and albula) and whitefish ( L. generosus) Pythium monospermum were rarely found. collected in November. Results indicated that the eggs of The highest number of straminipilous species were vendace and whitefish from the various lakes were a found in the water from the Biala River (most eutrophic), habitat for 15-20 and 15-22 different fungi, respectively. while the lowest number were found growing on the eggs Saprolegnia parasitica was found on vendace eggs from in containers with water from the Suprasl River (less only three lakes, while Aphanomyces laevis and S. eutrophic). The highest number of infected eggs of both parasitica occurred on whitefish eggs collected from all species was found in water from the Biała River (27.3% 10 lakes. A comparison of data of eggs obtained from

506 Afr. J. Microbiol. Res.

Table 2. Number of straminipilous organisms recorded on the eggs of Stenodus species in water of particular water bodies.

S. leucichthys (n = 750) S. nelma (n = 750) Taxa River Biała River Supraśl Pond Komosa River Biała River Supraśl Pond Komosa Straminipila

Peronosporomycetes (Oomycota)

Saprolegniales

Achlya bisexualis Coker et Couch 8 3 - - 2 4 A.dubia Coker

A.flagellata Coker 7 4 5 - - - A.hypogyna Coker et Pember. - - - 7 3 6 A.polyandra Hildebr. 5 2 3 - - - A.racemosa Hildebr. 18 4 7 14 4 6 A.radiosa Maurizio - - - - - 7 A.rodrigueziana F.T.Wolf 7 - 4 - - - A.treleaseana (Humphr.) Kauffm. - - 6 - - - Aphanomyces laevis de Bary 9 - - - - - A.frigidophilus Kitauch. et Hatai

Dictyuchus monosporus Leitgeb 10 - 5 - - - D. pisci Khulbe et Sati - - - 8 - 5 Isoachlya monilifera (de Bary) Kauff. - - - 11 7 - Leptolegnia caudata de Bary - - - 7 - - Saprolegnia australis R.F.Elliott 7 2 4 - - - S.diclina Humphrey

S.ferax (Gruith.)Thur. - - 8 - - - S.hypogyna (Pringsh.) de Bary - 4 7 - - - S.monoica Pringsh. - - - 8 - - S.parasitica Coker 21 8 12 24 11 14 S.polymorpha Willoughby - - - - - 8 S.salmonis Huss. et Hatai 6 - - - - - Leptomitales 46 14 26 38 20 27 Leptomitus lacteus (Roth) Agardh 11 - - - - - Pythiales - - - 7 11 - Pythium diclinum Tokun

P.inflatum V.D.Mathews - - - 19 - 7 P.monospermum Pringsh.

P.rostratum E.J.Butler

P.tenue Gobi 16 6 8 12 8 11 P. ultimum Trow - - - 7 - - 11 - - - - -

5 - - - - -

- - 6 - - -

13 - 4 - - -

Total number of species 20a 15b

Number of species in water from River Biała Number of species in water from River 16a 12a Supraśl Number of species in water from Pond Komosa 9b 8b

14c 10b

% of infected eggs 27.3a 6.3b 14.0c 21.6a 8.8b 12.7c

Means with the same letter are not significantly different (p>0.05).

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

Species of Amino acid Carbohydrate Urease genus Achlya Asp, Glu, Arg, Ala Fru, Glu, Man, Raf, Suc, Mal, Lac, Mel, Cel, Tre, Sta, Dex, Rha, Gly - Aphanomyces Glu, Ala, Cys Glu, Sta - Leptolegnia Asp, Glu, Ala Fru, Glu, Man, Mal, Mel, Cel, Tre, Sta, Dex, Gly + Pythium Ala, His Fru, Glu, Man, Gal, Raf, Suc, Mal, Lac, Mel, Cel, Tre, Sta, Dex, Rha, Gly, Sal + Saprolegnia Asp, Glu, Arg, Ala, His Fru, Glu, Man, Mal, Cel, Tre, Sta, Dex, Gly +

Amino acids = Ala, Alanine; Arg, arginine; Asp, asparagine; Cys, cysteine; Glu, glutamine; His, histidine. Carbohydrate = Fru, Fructose; Gal, galactose; Glu, glucose; Man, mannose; Mal, maltose; Mel, melibiose; Cel, cellobiose; Dex, dextrin; Gly, glycerol; Lac, lactose; Rha, rhamnose; Sal, salicin; Raf, rafinose; Sta, starch; Suc, sucrose; Tre, trehalose. ”+” positive; “-“ negative.

vendace and whitefish in 1994 and 1995 indicated genetic factor and resistance (Maule et al., 1989) of differences in the occurrence of fungi, possibly because salmonid populations to both bacterial diseases of different environmental conditions between the two (Beachman and Evelyn, 1992; Gjedrem and Gjöen, 1995) years that affected the condition of females during the and mycotic infections (Nilsson, 1992). Results of the prespawning period, egg quality, and/or the presence of investigations on the assimilation of amino acids, spores of particular fungal species. Environmental carbohydrates and urease by fungi are similar to records stressors considerably attenuate fish immunity, as in the for other salmonid fishes (Kitancharoen and Hatai, 1998; case of salmonid species (Maule et al., 1989). Czeczuga et al., 2011b) and for species from other Undoubtedly, the breeding of whitefish in tanks is a families (Yuasa and Hatai, 1996). significant stressor that reduces their immunological Straminipilous from the order Saprolegniales, status. For example, two species of trout, brook trout in especially Saprolegnia, Achlya, Aphanomyces and their natural habitat were more resistant to Saprolegnia Dictyuchus species, are responsible for the egg infection mycotic infections than pond-bred brown trout (Wald and in investigated Stenodus species. Amongst the Wizbach, 1992). Thus, the condition of females before Saprolegniales, Aphanomyces frigidophilus, Dictyuchus spawning can affect the quality of eggs (Maule et al., pisci, Saprolegnia polymorpha and Saprolegnia salmonis 1989). Stress leads to a reduction in the quality of the are rare species to fishes. A. frigidophilus and S. eggs, which become smaller and less resistant to salmonis were first described in Japan in salmonid fishes infections (Campbell et al., 1994). (Kitancharoen and Hatai, 1997; Hussein and Hatai, The immunity of a given population is also food 1999), and D. pisci was described from India (Khulbe, dependent (MacPhee et al., 1995). Sakai et al. (1992) 1994). S. polymorpha was first described by Willoughby found that chitin in trout food can stimulate fish (1998) in ornamental carp (Cyprinus carpio L. koi) in the resistance. The chitinous armour of crustaceans are United Kingdom. All four species occur in water bodies of known to be rich in carotenoids (Czeczuga et al., 2013), Poland. A. frigidophilus and S. salmonis have been found and interestingly, fewer fungi were found on the eggs of on the eggs of C. lavaretus, Salmo trutta L. (Czeczuga et vendace, which contain more carotenoids, than on eggs al., 2005a), and Salmo salar (Czeczuga et al., 2011a) from the same lake but with fewer carotenoids (Czeczuga and also on the eggs of some other sturgeon species and Muszyńska, 1998). This could explain the lesser (Czeczuga et al., 2011c). Dictyuchus pisci and S. number of fungi growing on the eggs of C. lavaretus, polymorpha have also been found on the eggs of some which contain more carotenoids (Czeczuga and sturgeonid species (Czeczuga et al., 2011c) and S. Muszyńska, 1998). Carotenoids may serve as a source of polymorpha in the muscles of Carassius auratus (L.) oxygen in low-oxygen environments, ensuring a better adults (Czeczuga et al., 2011d). state of the eggs and enhancing their resistance to All straminipilous species found on the Stenodus eggs mycotic infections (Karnaukhov, 1973). Salmonid species (except A. frigidophilus, D. pisci and S. salmonis, which are oxygen dependent, and eutrophic waters contain less have been described only as necrotrophic species) oxygen than water with a lower level of eutrophication. belong to an opportunistic pathogen, which is sapro- and Thompson et al. (1994) showed that diet containing necrotrophic. Some of these species can grow on vitamin A increases the resistance of Atlantic salmon specimens of cryptogams (Czeczuga et al., 2005b), (Salmo salar L.) individuals. Carotenoids are known to be whereas others can grow on seed plants of both gymno- the precursors of vitamin A. sperms and angiosperms. Representatives of opportu-

Studies have also investigated the significance of the nistic pathogens have been found on specimens of all macro-

508 Afr. J. Microbiol. Res.

Figure 1. Lifecycle of Saprolegnia parasitica. A- sexual cycle: 1- oogonium; 2- antheridium; 3- sexual process; 4- oogonial release; 5- oospore; 6- germinated oospore. B- asexual cycle: 1- sporangium with zoospores; 2- sporangial release; 3- primary zoospore; 4- primary cyst; 5- primary zoospore are connecting with primary cyst; 6- secondary zoospore (considered as a main dispersed and infectious spore of Saprolegnia species); 7- polyplanetism; 8- secondary cyst with boat hooks; 9- germinated cyst (infecting fishes).

phytes (Czeczuga et al., 2005b). S. parasitica was one of process called polyplanetism (Beakes, 1983). Secondary the most frequently occurring species on the examined cysts are an infective form for fishes. eggs of both Stenodus species. Investigations have been carried out on the The lifecycle of S. parasitica consists of a sexual and environmental factors influencing the zoospores from asexual phase (Figure 1). Hyphal cells form the sexual different Saprolegnia species (Smith et al., 1989), the structures of antheridia and oogonia, which release morphological features of the vegetative hyphae (Ali, motile oospores. After germination, these oospores 2005), and the ultrastructure of Saprolegnia secondary release a new mycelium. These sexual structures have zoospore cyst ornamentation (from infected wild brown been found in taxonomical investigations of several trout, indicating two distinct morphotypes amongst long- species of Saprolegniales (Dick, 2001). The asexual spined species) (Fregeneda-Grandes, 2000). The sporangia and spores are formed at the end of hyphae, morphology of the cyst of the first morphotype shows a and this releases many motile zoospores (Bruno and higher number of bundles with a greater number and Wood, 1999), which are the primary zoospores. These length of hair, whereas the cysts from the second group swim for a short time and encyst in secondary zoospores, have fewer bundles and less and shorter hair. Cysts of which are motile for a longer period and have been the saprolegniosis isolated from the salmonids in our considered to be the main infectious spores (van West, study belonged to the second group, whereas most 2006). After encystment, the cyst releases a new isolates from fish muscle and river water belonged to the secondary zoospore. The secondary cyst of Saprolegnia first group (Fregeneda-Grandes, 2000). In the genera species can possess hairs, which in S. parasitica contain Saprolegnia and Isoachlya, hair has been found on the the “boat hooks.” The connection between the secondary cyst of secondary zoospores (Pickering et al., 1979; zoospore and secondary cyst forms a new cyst, a Beakes, 1983; Hallet and Dick, 1986). Other studies

Czeczuga et al. 509

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