Saprolegnia diclina: another species responsible for the emergent disease 'Saprolegnia infections' in amphibians.

Many amphibians are known to suffer embryonic die-offs as a consequence of an emergent disease known as 'Saprolegnia infections'. Thus far, the only species of Saprolegnia shown to be involved in natural infections is Saprolegnia ferax. In this study, we have isolated and characterized another Saprolegnia species responsible for 'Saprolegnia infections' on embryos of Bufo calamita in mountainous areas of central Spain. The strain was identified as belonging to Saprolegnia diclina based on morphological, physiological and molecular characters (sequencing of the internal transcribed region of ribosomal DNA). Zoospores of the new strain were able to infect embryos of Bufo calamita, and the symptoms observed were the same as those observed in natural infections. The results presented emphasize the need to carry out isolations and characterizations of the species and/or strains involved in this emergent disease. This will be important in order to design strategies to prevent the impact and spread of species (or strains) pathogenic to amphibians.     

Re-evaluation of the enigmatic species complex Saprolegnia diclina-Saprolegnia parasitica based on morphological, physiological and molecular data

The phylogenetic relationships among isolates of the Saprolegnia diclina-Saprolegnia parasitica complex were investigated based on ITS rDNA sequences, and correlated with morphological and physiological characters. The isolates studied belong to five phylogenetically separate clades. The majority of presumed parasitic isolates, mostly isolated from fish lesions, fell within a clade that comprises isolates which has been variously named as S. diclina Type 1, S. parasitica, Saprolegnia salmonis or just as unnamed Saprolegnia sp. Presence of bundles of long-hooked hairs on secondary cysts, high frequency of retracted germination, and oogonia production at 7 degrees C (when occurring) were characteristic of this clade. A single isolate identified as S. diclina Type 2 clustered in a clade along with Saprolegnia ferax isolates. The isolates identified as S. diclina s. str. (S. diclina Type 3) distributed in two clades and appeared closely related to Saprolegnia multispora and to a number of Chilean isolates identified as Saprolegnia australis. The ITS sequences of clade I were almost identical even though the isolates were of diverse geographical origins and showed physiological and morphological differences and variations in their pathogenicity. This suggest these species reproduces clonally even in apparently sexually competent isolates. Adaptation to parasitism in Saprolegnia might have occurred at spore level by the development of long-hooked hairs to facilitate host attachment and selection of a retracting germination. The use of the name S. parasitica should be assigned to isolates of clade I that contained isolates forming cysts with bundles of long-hooked hairs.     

Fungi isolated from cultured eggs, alevins and broodfish of brown trout in a hatchery affected by saprolegniosis

The aquatic fungi cultured from eggs, alevins and broodfish of brown trout Salmo trutta belonged to the genus Saprolegnia and were identified as S. diclina , S. australis , S. ferax , S. furcata , S. hypogyna , S. unispora and S. parasitica . The species obtained from infected eggs and alevins were different to those from infected fish. Several Saprolegnia species were isolated from eggs and alevins, whereas all the isolates obtained from broodfish were the pathogenic S. parasitica .     

Mass mortality in cultured coho salmon (Oncorhynchus kisutch) due to Saprolegnia parasitica coker.

Epizootics of saprolegniasis occurred in 20 to 60 g freshwater-cultured coho salmon (Oncorhynchus kisutch) in Miyagi Prefecture, Japan. Cotton-like mycelia occurred on the body surface of infected fish, especially around the head, the adipose fin and the caudal fin, and aseptate hyphae occurred in the lesions. The hyphae also penetrated into the muscle and blood vessels. The isolated fungus was identified by asexual morphological characteristics as Saprolegnia parasitica (syn. S. diclina Type 1), a known salmonid fish pathogen.     

Developmental alterations and osmoregulatory physiology of a larval anuran under osmotic stress

Water salinity represents an environmental stress for many species. Amphibians are particularly sensitive because they are generally poor osmoregulators, and most species are completely absent from brackish and saline environments. We experimentally examined the effect of different salinity levels on larvae of the toad Bufo calamita L., a species that occupies freshwater ponds but can also breed in brackish ponds. Two independent experiments are reported here. In both experiments, tadpoles under saline conditions (ranging between 85 and 200 mOsm) showed a slower developmental rate, metamorphosing between 4 and 9 d later than the controls. Bufo calamita tadpoles reared in brackish water increased their osmolality and solute concentration (mainly sodium and chloride), decreased their levels of glucose, and decreased the total protein content, all measured from whole-animal extracts. Although most larval anurans are strictly ammoniotelic until the completion of metamorphosis, a few species exposed to dehydrating environments have evolved the ability to use urea as an osmolyte during the larval phase. The data presented here reveal that although B. calamita seems to be yet another exception to the rule of larval strict ammoniotelism, the tadpoles are not able to use urea as an osmolyte and rely on sodium-chloride balance instead. Preliminary immunoassays of thyroid hormone content suggest a possible decrease in hormone levels induced in water salinity conditions that correlate with a decreased developmental rate.     

Characterisation of Saprolegnia sp. isolates from channel catfish.

Nineteen channel catfish isolates of Saprolegnia sp. obtained from 5 separate fish farms in Mississippi, which became affected by winter kill syndrome during 1991 and 1996, were investigated with respect to physiological characteristics and genetic variation. Isolates of S. parasitica from crayfish and S. diclina were included for comparison. Most strains of catfish isolates grew well at 20 and 30 degrees C. Repeated zoospore emergence was found in catfish isolates of Saprolegnia sp. and S. parasitica, but not in S. diclina. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied for a further characterisation of the isolates. The RAPD analysis among Saprolegnia spp. isolates was constructed from 686 amplified products in 67 separable positions and indicated that the catfish isolates of Saprolegnia sp. are composed of 3 genetically distinct groups.     

Persistence of British natterjack toad Bufo calamita Laurenti (Anura: Bufonidae) populations despite low genetic diversity

Like other amphibians native to Britain, the natterjack toad Bufo calamita must have colonized the islands during the relatively short period between the end of the last glaciation and the separation of Britain from mainland Europe by rising sea levels. Unlike the other native amphibians, however, B. calamita is a habitat specialist at the north-westerly edge of its biogeographical range and for most of the 8000–10000 years since its colonization has probably been restricted to open dunes, heathlands and upper saltmarshes, as isolated populations in a few discrete areas of the country. We have investigated the genetic diversity and relatedness of six widely separated British natterjack populations by allozyme analysis, and shown that all have very low diversity (Overall P _95%= 2.7%, H = 0.004) by comparison with other anurans, including natterjack populations in mainland Europe and common frogs ( Rana temporaria , L) in Britain. Eighty percent of loci were fixed for the same allele in all six British natterjack populations and genetic differentiation between colonies was extremely low. The possible significance of these findings to the persistence of small isolated populations at range edges is discussed.     

Resistance of perch eggs to attack by aquatic fungi

In the distinctive gelatinous Perca fluviatilis egg mass, limited fungal growth by Aphanomyces and Saprolegnia spp. especially S. diclina , occurred within dead eggs but did not spread to adjacent live eggs. Perch eggs exposed to parasitic challenge by Saprolegnia parasitica , S. dieclina (type III) and S. ferax , under fluctuating temperature regimes replicating spring water temperatures, did not have significantly greater mortality than did unchallenged controls. The observations suggest that perch eggs have some anti-fungal properties which usually prevent the spread of fungus throughout the egg mass and that under normal spring temperatures there should be negligible ecological consequences of fungal infection in perch egg masses.     

Variability in timing of larval season in an amphibian community in SW Spain

Timing of larval periods of 9 species of amphibians (Hyla meridionalis, Pelobates cultripes, Discoglossus galganot, Bufo calamita, Bufo bufo, Rana perezi, Triturus marmoratus, Triturus boscat, Pleurodeles waltl) was followed in 7 different years Total larval season may last from autumn to summer, whereas in low rainy years it may last from late winter to summer In rainy years, two species are early breeders, starting to breed m autumn, six species commonly start in mid-winter and one species always breeds later, in spring The widest interannual variability m the onset and duration of larval season occurs in the earliest breeders In general, the more rainy the autumn and the higher temperatures were reached, the earlier the season started In contrast, duration of the season was not correlated with rainfall and temperature Interannual variability is interpreted as the possibility of adjusting the reproduction of the species to the availability of breeding habitats, constrained by physiological features of the species and interspecific relationships     

The effects of ambient pH on nitrogen excretion in early life stages of the American toad (Bufo americanus)

Acidification of breeding ponds has been identified as a potential threat to the survival and health of North American amphibian populations. The effects of acid exposure on ion and acid-balance are well known, but there is little information on how environmental water pH influences nitrogen balance in amphibians. The aim of this study was to determine the effects of moderately acidic water (pH 6.0) on nitrogen excretion in early life stages of the toad, Bufo americanus. Acid exposure (pH 6.0, 54 h) resulted in a 20–80% increase in ammonia-N excretion rates in embryos and early, middle and late tadpoles stages, whereas there was no significant effect on urea-N excretion. Tissue ammonia concentrations were significantly higher (+ 33%) in the embryos and 35–65% lower in the three groups of tadpoles exposed to water of pH 6.0 compared to control animals (pH 8.5). In embryos, ammonia excretion accounted for greater than 90% of total nitrogen excretion (ammonia-N + urea-N), but by the late tadpole stage this value had decreased to approximately 65%. These findings indicate that exposure of embryonic and larval B. americanus to moderately acidic water disrupts nitrogen balance by increasing nitrogen loss as ammonia, with no compensatory decrease in urea excretion.     

Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis

We assessed the diversity and phylogeny of Saprolegniaceae on amphibian eggs from the Pacific Northwest, with particular focus on Saprolegnia ferax, a species implicated in high egg mortality. We identified isolates from eggs of six amphibians with the internal transcribed spacer (ITS) and 5.8S gene regions and BLAST of the GenBank database. We identified 68 sequences as Saprolegniaceae and 43 sequences as true fungi from at least nine genera. Our phylogenetic analysis of the Saprolegniaceae included isolates within the genera Saprolegnia, Achlya and Leptolegnia. Our phylogeny grouped S. semihypogyna with Achlya rather than with the Saprolegnia reference sequences. We found only one isolate that grouped closely with S. ferax, and this came from a hatchery-raised salmon (Idaho) that we sampled opportunistically. We had representatives of 7-12 species and three genera of Saprolegniaceae on our amphibian eggs. Further work on the ecological roles of different species of Saprolegniaceae is needed to clarify their potential importance in amphibian egg mortality and potential links to population declines.     

Studies in aquatic fungi of varanasi. X. Additional new or interesting records of aquatic phycomycetes

Summary Four species of the genusSaprolegnia Nees, viz.,Saprolegnia diclina, S. delica, S. mixta, S. monoica var.glomerata, and three from the achlyoid group, viz.,Achlya colorata, A. racemosa, andA. americana were isolated on various substrata used as baits from the vicinity of University campus. ExcludingS. diclina andA. americana, all the above fungi are reported as occurring new from this country.     

Genetic variability in geographic populations of the natterjack toad (Bufo calamita)

Across altitudinal and latitudinal gradients, the proportion of suitable habitats varies, influencing the individual dispersal that ultimately can produce differentiation among populations. The natterjack toad (Bufo calamita) is distributed across a wide geographic range that qualifies the species as interesting for a geographic analysis of its genetic variability. Five populations of B. calamita in the Sierra de Gredos (Spain) were studied in an altitudinal gradient ranging from 750 to 2270 m using microsatellite markers. In addition, we analyzed the latitudinal genetic variation in B. calamita within a global European distribution using genetic diversity parameters (mean number of alleles per locus [M(a)] and expected heterozygosity [H(E)]) obtained from our results and those published in the literature. The low level of genetic differentiation found between populations of B. calamita (F(st) ranging from 0.0115 to 0.1018) and the decreases in genetic diversity with altitude (M(a) from 13.6 to 8.3, H(E) from 0.82 to 0.74) can be interpreted by the combined effects of discontinuous habitat, produced mainly by the high slopes barriers and geographic distance. In the latitudinal gradient, genetic diversity decreases from south to north as a consequence of the colonization of the species from the Pleistocene refugium. We conclude that the genetic variability in B. calamita along its wide altitudinal and latitudinal geographic distribution mainly reflects the colonization history of the species after the last glacial period.     

Patterns of somatic embryo formation in Siberian larch: embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5-1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5-10 days, such embryos formed the embryogenic tissue including two cell types--elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Some species of saprolegniaceae collected from Gorakhpur,India

Summary Eleven species of Saprolegniaceae viz., Saprolegnia diclina, S. ferax, Isoachlya anisospora, Achlya dubia, A. proliferoides, A. diffusa, A. orion, A. prolifera, A. conspicua, Aphanomyces helicoides, and Leptolegnia sp. (sterile) collected from waters of Gorakhpur have been described.This forms part of a thesis accepted for the degree of Ph. D. in the University of Gorakhpur, Gorakhpur.     

Patterns of somatic embryo formation in Siberian larch: Embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5–1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5–10 days, such embryos formed the embryogenic tissue including two cell types—elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

Water-soluble proteins in early amphibian embryos. III. Immunoelectrophoretic analysis of the antigenic changes in the ectoderm of the early gastrula and neural plate during development

20 water-soluble antigen have been identified with the help of rabbit antisera to extracts of the early gastrula ectoderm and neural plate in Rana temporaria. All of them were also found in the early blastula embryos and unfertilized eggs. The identified antigens are characterized by a definite embryospecificity. As the development proceeds, the concentration of these antigens in the embryonic tissues decreases until the complete disappearance of corresponding immunoelectrophoretic reactions. By this characteristic all antigens under study are subdivided into four groups: I--five antigens identified at the early developmental stages only (until hatching, stage 29); II--nine antigens present up to stages 33--35; III--three antigens followed up to stages 39--40 (well formed tadpole); IV--three antigens were found at all developmental stages under study up to stages 45--47. 11 out of 20 identified antigens have antigenic similarity with the proteins of blood serum of adult amphibians. Besides, the early gastrula ectoderm contains antigens similar with those of the brain of adult amphibians.     

Adaptation or exaptation? An experimental test of hypotheses on the origin of salinity tolerance in Bufo calamita

The natterjack toad (Bufo calamita) shows variation in embryonic and larval salinity tolerance across populations in southern Spain. However, its aquatic/terrestrial biphasic life cycle, together with remarkable differences in salinity tolerance between Spanish and UK freshwater populations suggest an alternative hypothesis to local adaptation. Drought resistance during the terrestrial phase and salinity tolerance during the aquatic phase are both related to osmotic stress tolerance, and if there were an association between them, one could have evolved as an exaptation from the other. To test such an association, we reared B. calamita juveniles from three populations known to differ genetically in their salinity tolerance, under either dry or humid conditions. Drought decreased growth rate, enhanced burying behaviour, and decreased foraging activity and efficiency. No significant population x treatment interaction was found for any variable, i.e. populations were equally affected by drought. These results do not support the hypothesis of a genetic association between salinity and drought tolerance.     

Developmental toxicity of aflatoxin B1 in the rodent embryo in vitro: contribution of exogenous biotransformation systems to toxicity

Aflatoxin B1 (AFB1) is a known carcinogen and developmental toxin in various species of mammals, birds, amphibians, and fish. AFB1 requires metabolic activation (biotransformation) to the 2,3-epoxide metabolite for carcinogenicity; however, it is unknown if biotransformation is a prerequisite for AFB1 embryotoxicity. Cultured day 10 rat embryos were exposed to AFB1 alone and AFB1 in the presence of cofactors and hepatic S9 fractions from adult male rats induced with either phenobarbital or 3-methylcholanthrene. Under these different culture conditions qualitatively similar patterns of malformation were seen in all embryos exposed to AFB1. At culture concentrations of 15 microM or greater, AFB1 produced abnormalities in neural tube development in a concentration-dependent manner. The presence of hepatic S9 fractions had no effect on the ability of AFB1 to produce dysmorphogenesis in vitro or on the spectrum of malformations elicited. However, the addition of hepatic S9 fractions did greatly enhance the embryolethality of AFB1. This enhancement was greater with phenobarbital- than 3-methylcholanthrene-preinduced hepatic S9 fractions. Our results suggest that separate chemical mediators may be responsible for the embryolethal and dysmorphogenic effects of AFB1 observed in day 10 rat embryos in vitro. We found that the embryolethality of AFB1, but not the dysmorphogenicity, could be greatly modulated by exogenous biotransformation.