Viable endospores of Thermoactinomyces vulgaris in lake sediments as indicators of agricultural history

Bacteria of the genus Thermoactinomyces form endospores with an extreme longevity in natural habitats. We isolated Thermoactinomyces sacchari from 9,000-year-old varved (annually laminated) sediment; thus, T. sacchari is probably one of the oldest known living organisms. More importantly, we tested and verified the hypothesis that there is a relationship between concentrations of dormant, viable endospores of T. vulgaris in lake sediments and the extent of agriculture in the catchments of the lakes. In surface sediments, low concentrations were recorded in forest lakes and the concentrations increased with increasing areas of cultivated land around the lakes. In varved sediment cores from three lakes, we found a temporal relationship between records of T. vulgaris endospores and the pollen of plants indicating agriculture. Endospores were very rare in sediments deposited before agriculture, ca. 1100 A.D. From then to between 1300 and 1700 A.D., a period with restricted cultivation, low but more regular rates of accumulation of endospores were recorded. High endospore accumulation rates were found with the subsequent agricultural expansion. This investigation confirms suggestions that this bacterium could be used as a paleoindicator for agricultural activity and be complementary to pollen analyses. Viable bacteria in continuous records of lake sediments are also potential material for evolutionary studies.     

Viable endospores of Thermoactinomyces vulgaris in lake sediments as indicators of agricultural history.

Bacteria of the genus Thermoactinomyces form endospores with an extreme longevity in natural habitats. We isolated Thermoactinomyces sacchari from 9,000-year-old varved (annually laminated) sediment; thus, T. sacchari is probably one of the oldest known living organisms. More importantly, we tested and verified the hypothesis that there is a relationship between concentrations of dormant, viable endospores of T. vulgaris in lake sediments and the extent of agriculture in the catchments of the lakes. In surface sediments, low concentrations were recorded in forest lakes and the concentrations increased with increasing areas of cultivated land around the lakes. In varved sediment cores from three lakes, we found a temporal relationship between records of T. vulgaris endospores and the pollen of plants indicating agriculture. Endospores were very rare in sediments deposited before agriculture, ca. 1100 A.D. From then to between 1300 and 1700 A.D., a period with restricted cultivation, low but more regular rates of accumulation of endospores were recorded. High endospore accumulation rates were found with the subsequent agricultural expansion. This investigation confirms suggestions that this bacterium could be used as a paleoindicator for agricultural activity and be complementary to pollen analyses. Viable bacteria in continuous records of lake sediments are also potential material for evolutionary studies.     

Biogeochemical changes in the sediments of Lake Cantara South,a saline lake in South Australia

Biogeochemical studies were undertaken of a 65-cm long sediment core from Lake Cantara South, South Australia. ¹⁴C determinations indicated that the sediments had been deposited over 2000 years. Changes with sediment depth in the concentration or ratio of the following were determined: (i) total organic carbon, total carbonate (inorganic) carbon, total sulfur, total carbon, total inorganic and organic sulfur, atomic C/N, and sulfate/chloride; (ii) n-alkanes; (iii) a highly branched isoprenoid alkane, and (iv) steroids. Interpretation of the changes with sediment depth indicated the nature of changes that took place when the system changed from a protected marine lagoon to an isolated (athalassic) saline lake. This change took place about 1000 years ago.     

Applications of a rapid endospore viability assay for monitoring UV inactivation and characterizing arctic ice cores

We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by L-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% +/- 3.8% and 48.9% +/- 4.5%, respectively), while only 27.8% +/- 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 +/- 19 germinable spores/ml and 369 +/- 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% +/- 9.3%), and the second core contained 131 +/- 4 germinable spores/ml and 162 +/- 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% +/- 8.8%), whereas only 2 CFU/ml were detected by culturing.     

A multiproxy palaeoecological record of Holocene lake sediments from the Rio Tapajós, eastern Amazonia

Two sediment cores up to 42 m in length were raised from the wide, deep, section of the lower Tapajós River, Amazonia, referred to as Lago Tapajós. These cores reveal a history of subtle environmental change that began with the formation of Lago Tapajós as sea level rose about 11,000 years ago. The sediments of the lake were deposited fairly quickly—at a rate of ca. 4 m per millennium and are uniformly fine grained, with low organic content. The fossil pollen record derived from these sediments reveals that forest surrounded this site throughout the Holocene. The largest change in the core took place between ca. 5500 and 4200 cal. years BP and reflects a transition from coarser to finer sediments. Coincident with the change in sediment is a slight transition in the pollen spectra with an increase in Poaceae abundance at the expense of the pioneer tree Cecropia cecropia. A tentative explanation is offered in which increased human activity, possibly spurred by climatic change, resulted in the formation of some local grasslands. However, despite the apparent actions of humans, there is no indication of basin-wide transformation of landscapes in this record. The Holocene persistence of forest as the dominant landscape matrix around Lago Tapajós is supported by low δ¹³C values and by the constant geochemistry and mineralogy of the lake sediments.     

Extraction and Purification of Pasteuria spp. Endospores

Pasteuria penetrans is an endospore-forming bacterial parasite of root-knot nematodes that has potential as a biological control agent. Biochemical investigations of P. penetrans are limited because of difficulty in obtaining large quantities of endospores free of plant debris and contaminating microorganisms. Our objective was to develop a technique for extraction and purification of P. penetrans endospores from root-knot nematodes. Tomato roots infected with Meloidogyne arenaria that was parasitized by P. penetrans were digested with cytolase. The nematode females along with plant debris were washed with a jet stream of water onto an 800-µm-pore sieve nested on a 250-µm-pore sieve. The materials retained on the 250-µm-pore sieve were centrifuged through a 20% sucrose solution. The resulting loose pellet fraction was collected on a 250-µm-pore sieve and then centrifuged through a 47% sucrose solution. Endospore-filled females were handpicked from the 47% sucrose pellicle fraction. Endospores were released by grinding the females with a glass tissue grinder. The endospores were then filtered through a nylon filter with 8-µm openings, collected by centrifugation, and subjected to buoyant density centrifugation in different media. Further purification by buoyant density centrifugation in a linear gradient of sodium diatrizoate resulted in a preparation of endospores free of debris. This additional step may be desirable for the further characterization of components unique to the endospores.     

Seasonal rhythmites from a lower pleistocene lake in northeastern Spain

Chemical, sedimentological, ostracod and palynological data were obtained in order to investigate the rhythmic alternation of grey and beige silty layers, with the occasional presence of sandy layers, from a small lacustrine deposits in Catalonia.The deposit was formed in a shallow meromictic and slightly saline lake. Occasional floods from a nearby river brought in detritic material.The alternation of the silty layers is seasonal: pollen and ostracods demonstrate that the beige layers were deposited during spring and the grey layers towards the end of spring and after it, until the next spring.Graded bedding, bioturbation structures and iron nodules found in the deposit are also examined for further information on the formation of the deposit.Pollen suggests an age for the deposit ranging between the Senezian I (probably representing the Waalian Stage, after Elhaï, 1969) and the Granada Interstadial (which is believed to correspond to the Holsteinian Stage by Florschütz et al., 1971).     

A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies

A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed, and filtered to collect large numbers of viable endospores. This method obviates laborious handpicking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations.     

Laminated Eocene maar-lake sediments from Eckfeld (Eifel region, Germany) and their short-term periodicities

The Eocene age Eckfeld/Eifel maar lake is interesting both because of the excellently preserved fossil remains and also for the laminated sediment sequence deposited under subtropical conditions. The latitude, however, has a higher seasonal insolation difference than comparable recent environments due to the northward shift of climatic zones during the Eocene. The overall aim of this investigation was to compare sediment types and lake development of a Tertiary (subtropical) maar lake with recent maar lakes, to discuss the origin of laminations, and search for their possible periodicities. The main sediment types, which were exposed during recent paleontological excavations, are laminated oilshales, partly laminated bituminous siltstones, and graded beds (mostly turbidites), which sometimes include laminated clay tops. Some deeper parts of the profile drilled in 1980, are dominated by diatomaceous laminites. The amount of organic carbon (around 8.5%) is comparable with recent lakes with low sedimentation rates. The high C/N ratios (25–30) of the Eckfeld sediments, as well as the fact that the highest C_org values are found in the graded beds, shows the importance of terrestrial plant debris for these lake sediments. Lamination of the oil shales was caused by the complex interrelationship of clastic supply (mineral and plant detritus), autochthonous organic deposition (mainly green algae), and early diagenetic siderite formation. The darker sublaminae represent the coarser clastic (organic and inorganic) input together with the autochthonous supply from the lake (green algae), whereas the lighter sublaminae are composed of settled fine-grained, mostly siliciclastic detritus derived from the probable yearly turnover of the density stratification of the lake. The thickness of the fine siliciclastic and organo-clastic layers, the morphology and palaeogeographical situation of the maar lake, and the occurrence of algal layers, strongly suggest that the laminated sediments are varves (although there is no definitive proof of this). Thickness variations from ca. 700 oil shale laminae were measured and computed. Time series analysis yielded at least one certain period of 5.5 yr, accepting that the laminae are indeed varves. The duration of the laminated lake sequence from Eckfeld maar, as deduced from sedimentation rates of the different sediment types is ca. 82,000 yr and, thus, within the typical life time of other small maar lakes.     

Detrital layers marking flood events in recent sediments of Lago Maggiore (N. Italy) and their comparison with instrumental data

1. A succession of 20 detrital layers was detected in five short cores from the Pallanza Basin in the western part of Lago Maggiore (Italy) by combining thin‐section analyses and high‐resolution micro‐X‐ray fluorescence (μ‐XRF) scanning techniques. The detrital layers range in thickness from 0.6 to 17.4 mm and appear most distinct in the upper 20–25 cm of each core, where eutrophication since the early 1960s resulted in the deposition of a dark, organic sediment matrix. 2. The age‐depth model of previously dated cores was transferred by precise intra‐basin correlation of distinct marker layers, thus providing a reliable chronology for the 20 detrital layers covering the time period 1965–2006. 3. All detrital layers are related to regional floods as recorded by short‐term lake level rises and peaks in discharge of the River Toce, the main tributary to the Pallanza Basin. 4. Detailed intra‐basin correlation of detrital layers allows us to distinguish river run‐off events from local erosion, as well as evaluate the relation between detrital layer thickness and flood amplitude. Massive clay accumulation on top of the thickest detrital layers might have affected lake ecology by attenuating light and influencing metabolic activity. 5. In the clastic‐dominated sediments deposited before 1965, detrital layers are less clearly discernible because of the predominantly clastic pelagic sediment matrix. The combination of thin‐section and μ‐XRF techniques, however, shows the potential to establish even longer flood layer time series from Lago Maggiore sediments.     

Comparison of in vivo and in vitro inactivation of endospores of <Emphasis Type="Italic">Rhinosporidium seeberi</Emphasis> following dapsone treatment

Rhinosporidiosis in humans and animals, caused by Rhinosporidium seeberi, can now be termed an emerging infective disease worldwide. The pharmacokinetics of dapsone when used as an antimicrobial agent in patients with rhinosporidiosis was compared with its pharmacodynamics in the inactivation of purified rhinosporidial endospores in vitro. A marked discrepancy was noted between the in vitro inactivation, which commenced on day 1 and was a maximum at day 4, whereas earlier reports on the response of endospores in patients under dapsone therapy indicated that the degeneration and disappearance of the endospores was not observed for 18–36 weeks after therapy began. Reasons for this discrepancy that operate in vivo are postulated: (a) impermeability of the barriers of down-growths of squamous epithelium in which the endospore-containing rhinosporidial sporangia are embedded; (b) presence of a mucoid matrix in which the endospores exist within the sporangia. These explanations contribute to resolving the controversial problem of general correlations between in vitro and in vivo results from studies on the action of antimicrobial drugs.     

A late pleistocene and holocene vegetation and environmental record from Lake Wangoom, Western Plains of Victoria, Australia

Pollen, microfaunal and sedimentological evidence from the top 20 m of sediment in a closed volcanic crater lake is used to construct a detailed record of vegetation and environmental conditions through the Holocene and a substantial part of the Late Pleistocene. Radiocarbon dating suggests that the sequence covers tha last 51,000 yr or so. High lake-levels and the presence of forest or woodland vegetation indicate that the Holocene and the basal few thousand years of the record experienced wet and warm conditions. The earliest period was succeeded by a long phase of lower but variable moisture levels before more arid conditions resulted in the replacement of forest and woodland by herbaceous vegetation and frequent lake drying. Periods of slight amelioration occurred between about 27,000 and 19,000 and before 15,000 radiocarbon yr ago. Maximum aridity occurred between about 19,000 and 10,000 yr B.P., a period incorporating the height of the last glacial. During the Pleistocene, the lake became gradually more saline but has been fresh during the Holocene, even under low lake-levels.

An increase in charcoal values is recorded, probably around 20,000 years ago, and this could have resulted from increased burning as a result of the activities of Aboriginal people. This may have also caused a change in understorey vegetation from one composed largely of Asteraceae, to grassland.

The record is compared with others from southeastern Australia and there is good correspondence through the last 20,000 or 30,000 yr but, before this, problems of dating and the condensed nature of most sequences prevent detailed correlation.     

A burial cave in the western Aleutian Islands, Alaska

During the 1998 field season, the Western Aleutians Archaeological and Paleobiological Project (WAAPP) team located a cave in the Near Islands, Alaska. Near the entrance of the cave, the team identified work areas and sleeping/sitting areas surrounded by cultural debris and animal bones. Human burials were found in the cave interior. In 2000, with permission from The Aleut Corporation, archaeologists revisited the site. Current research suggests three distinct occupations or uses for this cave. Aleuts buried their dead in shallow graves at the rear of the cave circa 1,200 to 800 years ago. Aleuts used the front of the cave as a temporary hunting camp as early as 390 years ago. Finally, Japanese and American military debris and graffiti reveal that the cave was visited during and after World War II. Russian trappers may have also taken shelter there 150 to 200 years ago. This is the first report of Aleut cave burials west of the Delarof Islands in the central Aleutians.     

Novel heliobacteria of a few semi-arid tropical soils

Out of forty rhizosphere soils collected from semi arid tropical regions of Andhra Pradesh, India, 30 gave positive enrichments for heliobacteria. These bacteria were recognized by the presence of bacteriochlorophyll-g together with endospores in the initial enrichments. Using group specific primers of 16S rRNA gene, ten monoheliobacterial cultures were sequenced. They were finally grouped into 3 clusters based on the 16S rRNA gene similarity. Based on a few phenotypic characters, in addition to genetic characterization, we identified them as potential novel species and the 16S rRNA gene sequences were deposited with EMBL.     

Presence of Hydrogenophilus thermoluteolus DNA in accretion ice in the subglacial Lake Vostok, Antarctica, assessed using rrs, cbb and hox

The 3561 m Vostok ice core sample originating from the subglacial Lake Vostok accretion (frozen lake water) ice with sediment inclusions was thoroughly studied by various means to confirm the presence of the thermophile bacterium Hydrogenophilus thermoluteolus reported earlier in the 3607 m accretion ice sample. PCR and molecular-phylogenetic analyses performed in two independent laboratories were made using different 16S rRNA gene (rrs) targeted primers. As a result, rrs-targeted PCR permitted to recover several very closely related clones with a small genetic distance to Hydrogenophilus thermoluteolus (< 1%). In addition, RubisCO (cbbL or rbcL) and NiFe-Hydrogenase (hoxV or hupL) targeted PCR have also allowed to recover sequences highly related to Hydrogenophilus thermoluteolus. All these results point to the presence of thermophilic chemoautotrophic microorganisms in Lake Vostok accretion ice. They presumably originate from deep faults in the bedrock cavity containing the lake in which episodes of seismotectonic activity would release debris along with microbial cells.     

Tooth growth in male Antarctic fur seals (Arctocephalus gazella) from South Georgia: an indicator of long-term growth history

Growth of upper canine teeth of male Antarctic fur seals ( Arctocephalus gazella ) which died of natural causes at Bird Island, South Georgia, was quantified from measurements of annual layers in longitudinal sections of teeth. Mean age at death was 7.69±0.07 years and this showed a small but significant increase through the period when samples were collected (1972/73–1988/89). There were significant correlations between morphometrics of teeth and those of seals, suggesting that tooth growth provided an indication of body growth. Tooth growth rate was lowest in seals which died early (age 4 years) and increased with age at death. Changes in the growth pattern of teeth suggested that fur seals which became sexually mature early also died early. Tooth growth layers deposited in each calendar year were compared with the expected layer depth based on a linear relationship between layer depth and age at which each layer was deposited. There was significant variation in the depth of tooth growth layers deposited in different years, suggesting that growth was greater in some years than others. No trends in cohort strengths were detected, but particularly poor years for growth were closely related to years in which reproductive performance was also observed to be low. Variations in growth from 1967/68 to 1987/88 were correlated significantly (P < 0.008) with the Southern Oscillation Index of climatic variation.     

Sedimentary features and the evolution of lake Honghu,central China

Palaeolimnological studies of the sediments of Honghu, a large shallow lake in Central China, were used to investigate the history of the origin, formation, and changing extent of the lake. The results indicate that Honghu is a naturally-dammed feature, formed about 3000 years ago by meandering of the Changjiang river. Lake level then fell in the period after 2500 BP, but during the Jin epoch (265–420 AD) it recovered, only to fall once more after the Song dynasty (969–1279 AD). The most recent episodes of reflooding are dated to ca. 400 years ago, and to the late nineteenth century.     

Chitinase genes in lake sediments of Ardley Island, Antarctica

A sediment core spanning approximately 1,600 years was collected from a lake on Ardley Island, Antarctica. The sediment core had been greatly influenced by penguin guano. Using molecular methods, the chitinolytic bacterial community along the sediment core was studied over its entire length. Primers targeting conserved sequences of the catalytic domains of family 18 subgroup A chitinases detected group A chitinases from a wide taxonomic range of bacteria. Using quantitative competitive PCR (QC-PCR), chitinase gene copies in each 1-cm section of the whole sediment column were quantified. QC-PCR determination of the chitinase gene copies indicated significant correlation with phosphorus and total organic carbon concentration, suggesting a historical connection between chitinase gene copies and the amount of penguin guano input into the lake sediment. Most of the chitinase genes cloned from the historic sediment core were novel. Analysis of the chitinase gene diversity in selected sediment layers and in the fresh penguin deposits indicated frequent shifts in the chitinolytic bacterial community over time. Sequence analysis of the 16S rRNA genes of chitinolytic bacteria isolated from the lake sediment revealed that the isolates belonged to Janthinobacterium species, Stenotrophomonas species of gamma-Proteobacteria, Cytophaga species of the Cytophaga-Flexibacter-Bacteroides group, and Streptomyces and Norcardiopsis species of Actinobacteria. Chitinase gene fragments were cloned and sequenced from these cultivated chitinolytic bacteria. The phylogeny of the chitinase genes obtained from the isolates did not correspond well to that of the isolates, suggesting acquisition via horizontal gene transfer.     

Holocene vegetation history of the southern Levant based on a pollen record from Lake Kinneret (Sea of Galilee), Israel

Lake Kinneret, also known as the Sea of Galilee and Lake Tiberias, is located in the northeast of Israel. At a lake level of 211 m b.s.l. (below mean sea level), the central basin is 43 m deep. The maximum length of the lake is 21 km (N–S) and its maximum width is 12 km (W–E). Lake Kinneret’s surface area is 166 km². A new 17.8 m long sedimentary core was drilled in 2010. Here, we present the entire palynological record from it, which covers the last?~?9,000 years. Special emphasis is given to the natural and human-influenced vegetation history of Galilee in comparison to that of the more southerly Dead Sea region. Significant signs of human impact are the first Olea (olive) increase during the beginning of the Chalcolithic period between 7,000 and 6,500 years ago, as well as the prominent Olea phase during the Hellenistic/Roman/Byzantine period between 2,300 and 1,500 years ago. Mediterranean macchia and bathas scrub vegetation, as known in the area today, has developed in the southern Levant under human impact since the last ca. 1,500 years.     

Use of the fluorescent probe LAURDAN to label and measure inner membrane fluidity of endospores of Clostridium spp

A method for measuring the fluidity of inner membranes of populations of endospores of Clostridium spp. with a fluorescent dye was developed. Cells of Clostridium beijerinckii ATCC 8260 and Clostridium sporogenes ATCC 7955 were allowed to sporulate in the presence of 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) on a soil-based media. Labeling of endospores with LAURDAN did not affect endospore viability. Removal of the outer membranes of endospores was done using a chemical treatment and confirmed using transmission electron microscopy (TEM). Two-photon confocal laser scanning microscopy (CLSM), and generalized polarization (GP) measurements were used to assess fluorescence of endospores. Lipid composition analysis of cells and endospores was done to determine whether differences in GP values are attributable to differences in membrane composition. Removal of the outer membranes of endospores did not significantly impact GP values. Decoated, labeled endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 exhibited GP values of 0.77±0.031 and 0.74±0.027 respectively. Differences in ratios of fatty acids between cells and endospores are unlikely to be responsible for high GP values observed in endospores. These GP values indicate high levels of lipid order and the exclusion of water from within inner membranes of endospores.