CELL FORM AND SURFACE PATTERNS IN CHROOMONAS and CRYPTOMONAS CELLS (CRYPTOPHYTA) AS REVEALED BY SCANNING ELECTRON MICROSCOPY1

Fifteen freshwater cryptomonad species were freeze-dried and examined with the scanning electron microscope. Surveys of cell surfaces revealed four general cell types. Chroomonas type cells lack a furrow but possess a shallow vestibular depression where the flagella are inserted. The presence of a gullet could not be detected. Cryptomonas spp. displayed three morphological types, all lacking gullets. The first type of Cryptomonas has a simple, shallow furrow with ridges that apparently can close to form a raphe but an oval opening or stoma remains at the posterior end and an opening from the vestibulum is formed at the anterior end. The second Cryptomonas type consists of a complex furrow with furrow ridges and folds that extend almost two-thirds of the cell length. A sloma is present in the central region of the closed furrow. The folds apparently can separate thereby exposing the underlying furrow. The third type of Cryptomonas possesses a simple, non-closing furrow. At the anterior end there is a vestibular ligule which extends from the dorsalleft side of the cell and covers the region of the vestibulum where the contractile vacuole discharges.     

Length–weight relationship of giant snakehead,Channa marulius and stinging catfish,Heteropneustes fossilis from the River Ganga,India

The present study describes the length–weight relationship of two commercially important freshwater fish species of India viz., Channa marulius and Heteropneustes fossilis collected from the Ganga River, during the period January 2009–December 2010.     

Ultrastructural evidence for the presence of secretory cells in the pineal parenchyma ofHeteropneustes fossilis

There is an extensive literature dealing with the study of indoles, especially serotonin and melatonin, but with considerably less emphasis on the cells and cell types involved in the synthetic process. In the present electron microscopical investigation of the pineal end vesicle ofHeteropneustes fossilis, pinealocytes have been revealed in the pineal parenchyma characterized with extensive synthetic apparatus viz., rough endoplasmic reticulum, free ribosomes, lipid droplets, mitochondria and Golgi bodies. Two sub-populations of the pinealocytes are easily distinguishable on the basis of electron opacity and the preponderance of one or other morphological profile: light cells and dark cells. Light cells represent the active phase of secretion while dark cells represent the storage and release phase of secretion. A neuroendocrine role for the pineal body inHeteropneustes fossilis is suggested which may be significant in view of the nocturnal habit of the fish. Dedicated to my father, Prof. C B L Srivastava     

Endosymbiotic bacteria associated with the intracellular green algae ofHydra viridis

Gram-negative bacteria 4.5–5.5 μm in length and 1.2 μm in diameter are found in gastrodermal cells of three stains of freshwater green hydras,Hydra viridis (Ohio and Carolina from North America, Jubilee strain from England). They are motile via single polar flagella. They were detected in live animals, Jensen stained material, and electron micrographic sections. Bacteria lose motility quickly upon release from hydra cells. Green hydras harbor strain-specific numbers of chlorellae in these cells. Other tissue types lack algae. The chlorella-hydra symbiosis can be disassociated and the partners grown separately; transfer of photosynthate from algae to hydra occurs. Here we report the presence of endocellular bacterial vesicles specifically associated with cells that contain the symbiotic chlorellae. No cells that contained algae and lacked bacteria were seen. Vesicles, especially conspicuous in sexually mature green hydras, are probably produced upon extrusion from the cell. They contain either algae and bacteria or bacteria alone and are often expelled to the surrounding medium via the coelenteron. Bacteria are absent in nerve cells, interstitial cells, nematocysts, mucous cells, sperm, and probably in most of the other cell types that lack algae. They are present in at least one cell type that lacked algae: the columnar ovarian cells. Bacteria were lost in “bleached” hydras, those induced to lose their algae by high intensity light in a solution of DCMU, a standard inhibitor of photosynthesis. They were absent in a fourth strain of green hydra (Connecticut Valley,H. viridis) and inH. fusca andH. littoralis, two freshwater nonsymbiotic hydras. All of the hydra lacking bacteria contain conspicuous lipid droplets in their cells. The presence of large numbers of bacteria has implications for interpretations of metabolic exchange between host and algal symbionts and for extrapolation of metabolic data from strain to strain ofH. viridis.     

Transfer cells in the vascular parenchyma of roots

Structural adaptations to increased transport activities were investigated in the cells of vascular parenchyma at the site of the lateral root junction, in non-stressed plant roots. Typical transfer cells were differentiated in dicotyledonousHelianthus tuberosus and in two different genotypes ofH. annuus, the cv. IBH166 and a decorative form. In the representatives of monocotyledonous, no structural adaptations occurred in the roots ofHordeum vulgare but small and rare cell wall protuberances were found in xylem and phloem ofZea mays inbred line VIR17. Some degree of cell wall labyrinth differentiation was seen in xylem and typical transfer cells were found in phloem of the roots of the maize hybrid CE380. The capability of vascular parenchyma to differentiate transfer cells did not depend on species, genotype, or on the growing conditions withHelianthus. On the other hand, the development of the structural adaptations in monocotyledonous representatives depended on both the species and the genotype. This capability may be linked with the taxonomic and evolutionary position of plant species.     

Lignicolous freshwater ascomycota from Thailand: 1.Ascotaiwania sawada and its anamorph stateMonotosporella

Single ascospore isolates ofAscotaiwania sawada gave rise to conidia of aMonotosporella species. Repeated isolations confirmed that theMonotosporella was the anamorph ofA. sawada. These findings are discussed and compared with the other known anamorph-teleomorph stages of freshwater fungi. This species ofMonotosporella is new to science but because it has not been found in nature it is referred to as theMonotosporella state ofA. sawada.     

The detection of fish pathogenVibrio anguillarum in water and fish using a species-specific DNA probe combined with membrane filtration

The marine bacteriumVibrio anguillarum causes disease in fish worldwide and is particularly devastating in aquaculture. Little is known about the ecology ofV. anguillarum in the environment and how this may relate to the pathogenicity of this organism. Combining membrane filtration and a species-specific DNA probe, culturableV. anguillarum cells were detected in water from three habitats and in chinook salmon (Onchorynchus tshawytscha) tissue samples. Results show that different marine habitats have a marked effect on cell numbers and that water temperature may play a role in the culturability and distribution ofV. anguillarum. Vibrio anguillarum was detected from the gills of salmon within 24 h of transfer of fingerlings from freshwater to seawater, with cell numbers reaching a concentration of 1.9 × 10² cells g^–1 tissue 28 days post transfer.Vibrio anguillarum cell numbers were low in the colon throughout the study, andV. anguillarum was not detected in healthy kidney samples. The methodology reported in this paper allows the accurate quantification of culturableV. anguillarum cells and has allowed a preliminary study of the ecology of this species.     

Sperm ultrastructure in two species of the polychaete genusHarmothoe (Polynoidae)

The structure of spermatozoa is described for two species of polynoid polychaete,Harmothoe imbricata andHarmothoe impar, from material fixed and examined by both scanning and transmission electron microscopy. The two species undergo spermiogenesis within discrete testes. The testis ofH. imbricata is shown to have a layer of epithelial cells which possess an outer cuticular layer and a microvillous inner surface. Spermatocytes of both species are spherical but there are marked differences in the shape and size of the spermatozoa of the two species.H. impar has a classical primitive spermatozoon with a rounded head (2 μm long) and a button-shaped acrosome. Fully differentiated spermatozoa ofH. imbricata are modified from the primitive form by having a long head (10 μm length) with a pointed acrosome about 6 μm in length. Spermatozoa ofH. imbricata have a ring of up to fourteen mitochondria around a centrally inserted flagellum at the posterior whereasH. impar has a ring of four or five spherical mitochondria. Spermiogenesis is well synchronised inH. imbricata but all developmental stages can be found simultaneously in the testis ofH. impar. The differences in sperm structure of the two species may be related to differences in breeding biology which are hitherto unknown.     

Test for allelopathic interactions between two marine microalgal species grown in intensive cultures

Both high pH and cell senescence are believed to lead to the production of toxic extracellular metabolites in freshwater microalgae. However, there was no evidence for allelopathic suppression of photosynthesis when filtrates of either of two marine microalgae,Phaeodactylum tricornutum andDunaliella tertiolecta, were mixed with whole cultures of the other species. This was true even when filtrate ofP. tricornutum sample was derived from a culture at high pH or from one in various stages of senescence. It is believed that the major factor leading to the dominance ofP. tricornutum in intensive outdoor cultures is the unique ability of this alga to tolerate pH levels above 9.5, not the allelopathic inhibition of competing species.     

On the cell wall composition of the apiculate yeasts

Summary Sonic oscillation was used for the purpose of obtaining clean, chemically intact cell walls. The rate of disruption was determined for cells ofHanseniaspora uvarum andSaccharomyces cerevisiae. The carbohydrate fractions of cell walls ofHanseniaspora uvarum, H. valbyensis, Kloeckera apiculata, Saccharomycodes ludwigii andSaccharmyces cerevisiae were shown to be similar. Chromatography of cell wall hydrolysates of all these species demonstrated that glucose and mannose were the only sugars present (in about equal amounts) besides traces of glucosamine. The cell walls ofH. uvarum contained 78.1 per cent carbohydrates, 7 per cent protein and approximately 0.05 per cent of chitin. Fractionation of the polysaccharides lead to a recovery of 83.3 per cent of the carbohydrates present (30.4 per cent glucan and 34.9 per cent mannan). Saccharomyces cerevisiae cell walls were found to have a carbohydrate content of 82.8 per cent, 6.5 per cent protein and a trace of chitin (0.04 per cent). Nadsonia elongata contained a relatively large amount of chitin (ca. 5 per cent) and lacked mannan in its cell walls. It was concluded thatHanseniaspora andSaccharomycodes are closely related to theSaccharomyceteae but they have little in common with species ofNadsonia.     

The Toxic Symbiont Caedibacter caryophila in the Cytoplasm of Paramecium novaurelia

Endosymbiotic bacteria were observed to inhabit the cytoplasm of the freshwater ciliateParamecium novaurelia. Transmission electron microscopy and toxicity tests with sensitive paramecia showed that the endosymbionts belong to the genusCaedibacter. The bacteria conferred a killer trait to their host paramecia. The production of a proteinaceous inclusion body (“R-body”) in the bacterial cell makes them toxic to other paramecia after they become enclosed in food vacuoles. R-bodies ofCaedibacter sp were associated with phages, which are known in most otherCaedibacter species to code for the R-body proteins. The killer-effect ofP. novaurelia on sensitiveP. caudatum strains was of the “paralysis” type, which is a characteristic of the symbiont speciesCaedibacter caryophila. Until nowC. caryophila was known to inhabit the macronucleus ofParamecium caudatum only. Sequencing of the 16S rRNA-gene proved thatCaedibacter sp from the cytoplasm ofP. novaurelia belongs to the speciesC. caryophila as well. The rDNA-sequence of 1695 bp length differed in a total of only 1 bp from the corresponding gene inC. caryophila from the macronucleus ofP. caudatum. The results indicate that the infection of specific host cell compartments may depend on host genes, but not on different traits of the infecting symbiont species. The occurrence of killer and sensitive paramecia strains together in one pond is discussed with respect to the competitive advantage of the killer trait.     

Detection of the endangered European weather loach (Misgurnus fossilis) via water and sediment samples: Testing multiple eDNA workflows

The European weather loach (Misgurnus fossilis) is classified as highly endangered in several countries of Central Europe. Populations of M. fossilis are predominantly found in ditches with low water levels and thick sludge layers and are thus hard to detect using conventional fishing methods. Therefore, environmental DNA (eDNA) monitoring appears particularly relevant for this species. In previous studies, M. fossilis was surveyed following eDNA water sampling protocols, which were not optimized for this species. Therefore, we created two full factorial study designs to test six different eDNA workflows for sediment samples and twelve different workflows for water samples. We used qPCR to compare the threshold cycle (C_t) values of the different workflows, which indicate the target DNA amount in the sample, and spectrophotometry to quantify and compare the total DNA amount inside the samples. We analyzed 96 water samples and 48 sediment samples from a pond with a known population of M. fossilis. We tested several method combinations for long‐term sample preservation, DNA capture, and DNA extraction. Additionally, we analyzed the DNA yield of samples from a ditch with a natural M. fossilis population monthly over one year to determine the optimal sampling period. Our results showed that the long‐term water preservation method commonly used for eDNA surveys of M. fossilis did not lead to optimal DNA yields, and we present a valid long‐term sample preservation alternative. A cost‐efficient high salt DNA extraction led to the highest target DNA yields and can be used for sediment and water samples. Furthermore, we were able to show that in a natural habitat of M. fossilis, total and target eDNA were higher between June and September, which implies that this period is favorable for eDNA sampling. Our results will help to improve the reliability of future eDNA surveys of M. fossilis.     

Surface ultrastructure of the olfactory rosette of an air-breathing catfish,Heteropneustes fossilis (Bloch)

The surface architecture of the olfactory rosette ofHeteropneustes fossilis (Bloch) has been studied by scanning electron microscopy. The olfactory rosette is an oval structure composed of a number of lamellae arranged pinnately on a median raphe. The raphe is invested with epithelial cells and pits which represent goblet cell openings. On the basis of cellular characteristics and their distribution the lateral surface of each olfactory lamella is identified as sensory, ciliated non-sensory and non-ciliated non-sensory epithelium. The sensory epithelium is provided with receptor and supporting cells. The ciliated non-sensory epithelium is covered with dense cilia obscuring the presence of other cell types. The non-ciliated non-sensory epithelium is with many polygonal areas containing cells.     

Cluster analysis of genes for nitrogen fixation from several diazotrophs

Hierarchical clustering and similarity coefficients of pairwise alignments of the published nucleotide sequences of 27nifH genes suggest thatnif genes are as ancient as the archaebacteria and clostridia. The positions ofnifHl ofMethanococcus thermolithotrophicus, nifH3 ofClostridium pasteurianum, nifH3 ofAzotobacter vinelandii andnifH ofFrankia suggest that a variety of lateral transfers may have occurred during evolution ofnifH gene. The genes for type 3 nitrogenase ofA. vinelandii may have diverged early from methanogens and clostridia. A high similarity coefficient with the derived amino acid sequence of type 3 nitrogenase suggests the presence of a functionally similar enzyme inC. pasteurianum. The type 2 nitrogenase genenifH2 of azotobacters seems to have originated recently from the genenifHl for conventional type I nitrogenase. RhizobialnifH genes comprise two closely related but discrete clusters that are in consonance with the plasmid or chromosomal location ofnif genes. The chromosomal and plasmid locatednifH of rhizobia seem to have evolved independently but contemporaneously.     

Freshwater Euplotes Species with a 9 Type 1 Cirrus Pattern Depend upon Endosymbionts1,2

The hypothesis is advanced that all freshwater Euplotes species with a 9 type 1 fronto-ventral cirrus pattern (E. patella type) depend upon bacteria-like endosymbionts. Aposymbiotic cells of these species are unable to divide. The hypothesis is based on the investigation of 40 different freshwater Euplotes stocks collected in Germany, France, the USA, and Japan. No symbionts were found in E. crenosus and E. palustris, freshwater species with 10 fronto-ventral cirri, nor in E. muscicola, a representative of the freshwater Euplotes group with a 9 type 2 fronto-ventral cirrus pattern (E. affinis type). Characteristic for the essential endosymbionts are multiple nucleoids, a feature described earlier for omikron, an indispensable symbiont of E. aediculatus. Although the symbionts differ from omikron and among each other in size, shape, and their average number per host, they are believed to be related to omikron. In two stocks a different type of bacterium was found in which no defined nucleoids can be detected. Transfer of this symbiont into aposymbiotic cells, originally carrying omikron, revealed that it can restore the ability to multiply. Similarly, omikron was also able to restore the ability to divide in cells freed of this symbiont. It is assumed that this different type of symbiont is a secondary invader of Euplotes which displaced the original omikron-like endosymbiont. Some of the stocks were found to carry, in addition to omikron-like symbionts, other symbiotic bacteria; E. daidaleos carries in addition an alga. The findings suggest that the freshwater Euplotes species with a 9 type 1 cirrus pattern are closely related to each other and evolved from an ancestor (probably of cirrotype 10) which already was dependent upon endosymbionts of the omikron type. It supports the view that the two subgroups of freshwater Euplotes forms with a cirrotype of 9 have evolved independently from each other from species with 10 fronto-ventral cirri by losing a cirrus at different positions.     

Scanning and transmission electron microscopy of the squamose gill-filament epithelium from fresh- and seawater adaptedTilapia

Synopsis The architecture of the gill structure of variousTilapia species was studied in relation to their adaptability to hypersaline media. Using SEM and EM, it was shown that the squamose epithelial cells of the gills have species-typical patterns of ridges on their outer surfaces. These have previously been misinterpreted by other authors as microvilli or stereocillia. The ridges are more dense and better developed in euryhaline species, likeT. zillii, and less so in stenohaline species likeSarotherodon niloticus. Comparing freshwater and seawater-adapted individuals ofT. zillii, S. niloticus, S. galflaeus, andTristramella sacra, it was shown that in fresh water the surface cells are slightly swollen, extending over the openings of the chloride cells. During adaptation to sea water, these ridges become higher and denser and the cell surface shrinks, exposing the underlying orifices of the apical crypts of the chloride cells. The more euryhaline the species, the less change there is in the ridge pattern of the cells during passage from fresh to sea water. This evidence implicates the gill epithelium, together with the chloride cells, in the process of osmoregulation.     

Fine structure of the male reproductive system in two species ofHaplognathia sterrer (Gnathostomulida,Filospermoidea)

Summary The structure of the male reproductive systems of two species ofHaplognathia cf.lyra andH. cf.rosacea was described. The structure of the testes and the anterior portions of the sperm ducts in both species was found to be similar. However, considerable species differences were found between the structures of the glands and muscles associated with the reproductive systems. These were more elaborate inH. cf.lyra than inH. cf.rosacea. The former species possessed an H-shaped sperm duct gland, three distinct groups of penis muscles and a penis with two cell types and with a lumen. The latter species had paired sperm duct glands, no specialized penis muscles and a penis with only one cell type and without a detectable lumen. No open gonopore was observed in either species. The sperm presumably exit through a ventral tissue connection observed connecting the penis and the ventral epidermis. These findings were discussed in the light of Mainitz's (1977) theory concerning the primitive penis type within the Gnathostomulida.Abbreviations ap anterior-posterior penis muscles - bm basement membrane - csd common sperm duct - dl dorsal lumen of the penis - dp dorsal gland cells of the penis - dv dorsoventral muscles anterior to the penis - dw sperm duct wall cell - e epidermis - ex exit cell - g intestine - gl gut lumen - n nerve - p penis - sd sperm duct - sdg sperm duct gland - tw testes wall cell - vl ventral lumen of the penis - vp ventral gland cells of the penis This project was supported by NSF grant #GB 42211 (R.M. Rieger P.I.). The line drawings have been executed after our design by Ms. Linda McVay     

Viability ofSalmonella species in natural waters

The viability of different species ofSalmonella in seawater was studied under controlled laboratory conditions. The negative influence of water self-purifying factors exerted on the survival of the microorganisms varied depending on the factor considered and also on the specie selected. Biotic and luminous factors were mainly responsible for the inactivation and disappearance ofSalmonella in the water solutions. The addition of 1% of sewage to filtered seawater and freshwater provoked a decrease in the number ofSalmonella populations tested in comparison with the solutions without sewage. On the other hand, an increase of sublethally injuredSalmonella cells with respect to exposure time was not observed. From the results obtained from T₉₀ inactivation experiments, it may be deduced that the main factor implicated in the disappearance ofSalmonella from the water mass is physical dilution.     

Ecology ofVibrio cholerae in the freshwater environs of Calcutta,India

Seasonal incidence ofVibrio cholerae was monitored for a year in a man-made freshwater lake, an open sewage canal, and a pond composed of rainwater accumulations, located in Calcutta.V. cholerae was found in all sites. It exhibited a distinct bimodal seasonal cycle in the lake with a primary peak in August–September and a secondary peak in May–June. Correlation with environmental parameters revealed that temperature and, to a certain extent, pH were the important factors governing the densities ofV. cholerae. In the lake, sediment samples harbored high densities ofV. cholerae immediately after months when peak counts were observed in plankton, suggesting a cycle of cells between sediment and water. At the other sampling areas, no defined seasonality was observed. Instead, high counts ofV. cholerae were observed at these severely polluted sites throughout the study period, including the winter months. All the 15 water samples passed via the ligated loop of rabbits yielded pure cultures ofV. cholerae, indicating that the rabbit intestine selects outV. cholerae from a mixed flora. Uniformly high isolation rates ofV. cholerae were observed from brackish water and freshwater species of export quality prawns.V. cholerae was found to be abundant and was represented by 32 individual Louisiana State University (LSU) serovars, including two new serovars. The 01 serovar could not be isolated from any of the samples examined in this study. It was concluded thatV. cholerae non-01 is common in the freshwater environs of Calcutta.     

The effect of heated effluent on the occurrence and the reproduction of the freshwater limpetsAncylus fluviatilis Müller, 1774,Ferrissia wautieri (Mirolli, 1960) andAcroloxus lacustris (L., 1758) in two Dutch water bodies

The discharge of cooling water favours the occurrence of freshwater limpets. Reproduction ofAncylus fluviatilis is stimulated by the higher temperature of the cooling water; the life cycle ofFerrissia wautieri (nearly exclusively found in the outlet areas) does not, however, differ from that under natural temperature regimes. ForAcroloxus lacustris this is not clear as detailed studies on the life cycle under natural temperature circumstances are lacking. Acroloxus lacustris andFerrissia wautieri could be found in large numbers on hard substrates (stones), as well as on plant material in sheltered as well as in exposed situations. In this respect they differ fromAncylus fluviatilis, which is restricted in its occurrence to hard substrates and exposed situations.It is not very clear whether the occurrence ofAncylus fluviatilis is also influenced by some form of competition for food byBithynia tentaculata; both feed on diatoms. Further experimental studies of this question should yield interesting results.The changes in numbers of the three freshwater limpet species are mainly due to reproduction and migration, both largely governed by temperature and day length.It was possible to reconstruct the influence of cooling water in the Bergumermeer area by means of the ratio between the numbers ofAcroloxus lacustris andFerrissia wautieri. The share ofFerrissia was highest near the outiet and decreased with the distance from this station.