Geochemically Defined Space-for-Time Transects Successfully Capture Microbial Dynamics Along Lacustrine Chronosequences in a Polar Desert.

The space-for-time substitution approach provides a valuable empirical assessment to infer temporal effects of disturbance from spatial gradients. Applied to predict the response of different ecosystems under current climate change scenarios, it remains poorly tested in microbial ecology studies, partly due to the trophic complexity of the ecosystems typically studied. The McMurdo Dry Valleys (MDV) of Antarctica represent a trophically simple polar desert projected to experience drastic changes in water availability under current climate change scenarios. We used this ideal model system to develop and validate a microbial space-for-time sampling approach, using the variation of geochemical profiles that follow alterations in water availability and reflect past changes in the system. Our framework measured soil electrical conductivity, pH, and water activity in situ to geochemically define 17 space-for-time transects from the shores of four dynamic and two static Dry Valley lakes. We identified microbial taxa that are consistently responsive to changes in wetness in the soils and reliably associated with long-term dry or wet edaphic conditions. Comparisons between transects defined at static (open-basin) and dynamic (closed-basin) lakes highlighted the capacity for geochemically defined space-for-time gradients to identify lasting deterministic impacts of historical changes in water presence on the structure and diversity of extant microbial communities. We highlight the potential for geochemically defined space-for-time transects to resolve legacy impacts of environmental change when used in conjunction with static and dynamic scenarios, and to inform future environmental scenarios through changes in the microbial community structure, composition, and diversity.

Microbial Mats of the McMurdo Dry Valleys, Antarctica: Oases of Biological Activity in a Very Cold Desert.

Cyanobacterial mats in the Antarctic Dry Valleys are photosynthetic microbial ecosystems living at the extreme of conditions on Earth with respect to temperature, light, water and nutrient availability. They are metabolically active for about 8 weeks during the austral summer when temperatures briefly rise above freezing and glacial and lake melt waters are available. There is much to learn about the biogeochemical impact of mats in these environments and the microbial communities associated with them. Our data demonstrate that these mats attain surprisingly high rates of carbon (CO2) and dinitrogen (N2) fixation when liquid water is available, in some cases comparable to rates in warmer temperate or tropical environments. C and N2 fixation in Dry Valley mats in turn substantially elevate dissolved organic C and inorganic N pools and thereby promote enhanced microbial secondary production. Moreover, the microbial community fingerprint of these mats is unique compared with the more ubiquitous dry soils that do not contain mats. Components of the heterotrophic microbiota may also contribute substantially to N inputs through N2 fixation.

Rapid Microbial Dynamics in Response to an Induced Wetting Event in Antarctic Dry Valley Soils.

The cold deserts of the McMurdo Dry Valleys (MDV), Antarctica, host a high level of microbial diversity. Microbial composition and biomass in arid vs. ephemerally wetted regions are distinctly different, with wetted communities representing hot spots of microbial activity that are important zones for biogeochemical cycling. While climatic change is likely to cause wetting in areas not historically subject to wetting events, the responses of microorganisms inhabiting arid soils to water addition is unknown. The purpose of this study was to observe how an associated, yet non-wetted microbial community responds to an extended addition of water. Water from a stream was diverted to an adjacent area of arid soil with changes in microbial composition and activities monitored via molecular and biochemical methods over 7 weeks. The frequency of genetic signatures related to both prokaryotic and eukaryotic organisms adapted to MDV aquatic conditions increased during the limited 7 week period, indicating that the soil community was transitioning into a typical "high-productivity" MDV community. This work is consistent with current predictions that MDV microbial communities in arid regions are highly sensitive to climate change, and further supports the notion that changes in community structure and associated biogeochemical cycling may occur much more rapidly than predicted.

Further characterization of glycine-containing microcystins from the McMurdo dry Valleys of Antarctica.

Microcystins are hepatotoxic cyclic peptides produced by several cyanobacterial genera worldwide. In 2008, our research group identified eight new glycine-containing microcystin congeners in two hydro-terrestrial mat samples from the McMurdo Dry Valleys of Eastern Antarctica. During the present study, high-resolution mass spectrometry, amino acid analysis and micro-scale thiol derivatization were used to further elucidate their structures. The Antarctic microcystin congeners contained the rare substitution of the position-1 á´…-alanine for glycine, as well as the acetyl desmethyl modification of the position-5 Adda moiety (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid). Amino acid analysis was used to determine the stereochemistry of several of the amino acids and conclusively demonstrated the presence of glycine in the microcystins. A recently developed thiol derivatization technique showed that each microcystin contained dehydrobutyrine in position-7 instead of the commonly observed N-methyl dehydroalanine.

Intracellular immunohistochemical detection of tetrodotoxin in Pleurobranchaea maculata (Gastropoda) and Stylochoplana sp. (Turbellaria).

Tetrodotoxin (TTX), is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring.

No evidence for a culturable bacterial tetrodotoxin producer in Pleurobranchaea maculata (Gastropoda: Pleurobranchidae) and Stylochoplana sp. (Platyhelminthes: Polycladida).

Tetrodotoxin (TTX) is a potent neurotoxin found in the tissues of many taxonomically diverse organisms. Its origin has been the topic of much debate, with suggestions including endogenous production, acquisition through diet, and symbiotic bacterial synthesis. Bacterial production of TTX has been reported in isolates from marine biota, but at lower than expected concentrations. In this study, 102 strains were isolated from Pleurobranchaea maculata (Opisthobranchia) and Stylochoplana sp. (Platyhelminthes). Tetrodotoxin production was tested utilizing a recently developed sensitive method to detect the C9 base of TTX via liquid chromatography-mass spectrometry. Bacterial strains were characterized by sequencing a region of the 16S ribosomal RNA gene. To account for the possibility that TTX is produced by a consortium of bacteria, a series of experiments using marine broth spiked with various P. maculata tissues were undertaken. Sixteen unique strains from P. maculata and one from Stylochoplana sp. were isolated, representing eight different genera; Pseudomonadales, Actinomycetales, Oceanospirillales, Thiotrichales, Rhodobacterales, Sphingomonadales, Bacillales, and Vibrionales. Molecular fingerprinting of bacterial communities from broth experiments showed little change over the first four days. No C9 base or TTX was detected in isolates or broth experiments (past day 0), suggesting a culturable microbial source of TTX in P. maculata and Stylochoplana sp. is unlikely.

High levels of structural diversity observed in microcystins from Microcystis CAWBG11 and characterization of six new microcystin congeners.

Microcystins (MCs) are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Differences in the coding of the non­ribosomal peptide synthetase/polyketide synthase enzyme complex responsible for microcystin production have resulted in more than 100 microcystin variants being reported to date. The microcystin diversity of Microcystis CAWBG11 was investigated using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-mass spectrometry. This revealed that CAWBG11 simultaneously produced 21 known microcystins and six new congeners: [Asp3] MC-RA, [Asp3] MC-RAba, [Asp3] MC-FA, [Asp3] MC-WA, MC-FAba and MC-FL. The new congeners were putatively characterized by tandem mass spectrometry and chemical derivatization. A survey of the microcystin congeners produced by 49 cyanobacterial strains documented in scientific literature showed that cyanobacteria generally produce four microcystin congeners, but strains which produce up to 47 microcystin congeners have been reported. Microcystis CAWBG11 (which produces at least 27 congeners) was positioned in the top ten percentile of the strains surveyed, and showed fluidity of the amino acids incorporated into both position two and position four.

High-level diversity of tailed phages, eukaryote-associated viruses, and virophage-like elements in the metaviromes of antarctic soils.

The metaviromes of two distinct Antarctic hyperarid desert soil communities have been characterized. Hypolithic communities, cyanobacterium-dominated assemblages situated on the ventral surfaces of quartz pebbles embedded in the desert pavement, showed higher virus diversity than surface soils, which correlated with previous bacterial community studies. Prokaryotic viruses (i.e., phages) represented the largest viral component (particularly Mycobacterium phages) in both habitats, with an identical hierarchical sequence abundance of families of tailed phages (Siphoviridae > Myoviridae > Podoviridae). No archaeal viruses were found. Unexpectedly, cyanophages were poorly represented in both metaviromes and were phylogenetically distant from currently characterized cyanophages. Putative phage genomes were assembled and showed a high level of unaffiliated genes, mostly from hypolithic viruses. Moreover, unusual gene arrangements in which eukaryotic and prokaryotic virus-derived genes were found within identical genome segments were observed. Phycodnaviridae and Mimiviridae viruses were the second-most-abundant taxa and more numerous within open soil. Novel virophage-like sequences (within the Sputnik clade) were identified. These findings highlight high-level virus diversity and novel species discovery potential within Antarctic hyperarid soils and may serve as a starting point for future studies targeting specific viral groups.

Investigating diet as the source of tetrodotoxin in Pleurobranchaea maculata.

The origin of tetrodotoxin (TTX) is highly debated; researchers have postulated either an endogenous or exogenous source with the host accumulating TTX symbiotically or via food chain transmission. The aim of this study was to determine whether the grey side-gilled sea slug (Pleurobranchaea maculata) could obtain TTX from a dietary source, and to attempt to identify this source through environmental surveys. Eighteen non-toxic P. maculata were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 h, 24 h, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using liquid chromatography-mass spectrometry. Tetrodotoxin was detected in all organs/tissue after 1 h with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). Benthic surveys at sites with dense populations of toxic P. maculata detected very low or no TTX in other organisms. This study demonstrates that P. maculata can accumulate TTX through their diet. However, based on the absence of an identifiable TTX source in the environment, in concert with the extremely high TTX concentrations and short life spans of P. maculata, it is unlikely to be the sole TTX source for this species.

Structural characterization of new microcystins containing tryptophan and oxidized tryptophan residues.

Microcystins are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Eight of the (more than) 90 microcystin variants presently characterized, contain the amino acid tryptophan. The well-researched oxidation products of tryptophan; kynurenine, oxindolylalanine, and N-formylkynurenine, have been previously identified in intact polypeptides but microcystin congeners containing oxidized tryptophan moieties have not been reported. Liquid chromatography-tandem mass spectrometric analysis of an extract of Microcystis CAWBG11 led to the tentative identification of two new tryptophan-containing microcystins (MC­WAba and MC-WL), as well as eight other microcystin analogs containing kynurenine, oxindolylalanine and N­formylkynurenine (Nfk). Investigation of one of these congeners (MC­NfkA) by nuclear magnetic resonance spectroscopy was used to verify the presence of Nfk in the microcystin. Liquid chromatography-mass spectrometry analysis of a tryptophan oxidation experiment demonstrated that tryptophan-containing microcystins could be converted into oxidized tryptophan analogs and that low levels of oxidized tryptophan congeners were present intracellularly in CAWBG11. MC-NfkR and MC-LNfk were detected in standards of MC-WR and MC-LW, indicating that care during storage of tryptophan-containing microcystins is required.

Micro-eukaryotic diversity in hypolithons from miers valley, antarctica.

The discovery of extensive and complex hypolithic communities in both cold and hot deserts has raised many questions regarding their ecology, biodiversity and relevance in terms of regional productivity. However, most hypolithic research has focused on the bacterial elements of the community. This study represents the first investigation of micro-eukaryotic communities in all three hypolith types. Here we show that Antarctic hypoliths support extensive populations of novel uncharacterized bryophyta, fungi and protists and suggest that well known producer-decomposer-predator interactions may create the necessary conditions for hypolithic productivity in Antarctic deserts.

Tetrodotoxin concentrations in Pleurobranchaea maculata: temporal, spatial and individual variability from New Zealand populations.

Tetrodotoxin (TTX) is a potent neurotoxin that has been identified in a range of phylogenetically unrelated marine and terrestrial organisms. Tetrodotoxin was recently detected in New Zealand in Pleurobranchaea maculata (the grey side-gilled sea slug). From June 2010 to June 2011 wild specimens were collected from 10 locations around New Zealand. At one site (Narrow Neck Beach, Auckland) up to 10 individuals were collected monthly for 6 months. Attempts were also made to rear P. maculata in captivity. Tetrodotoxin was detected in samples from eight of the ten sites. The highest average (368.7 mg kg⁻¹) and maximum (1414.0 mg kg⁻¹) concentrations were measured in samples from Illiomama Rock (Auckland). Of the toxic populations tested there was significant variability in TTX concentrations among individuals, with the highest difference (62 fold) measured at Illiomama Rock. Tetrodotoxin concentrations in samples from Narrow Neck Beach varied temporally, ranging from an average of 184 mg kg⁻¹ in June 2010 to 17.5 mg kg⁻¹ by December 2010. There was no correlation between TTX levels and mass. The highest levels correspond with the egg laying season (June-August) and this, in concert with the detection of high levels of TTX in eggs and early larval stages, suggests that TTX may have a defensive function in P. maculata. Only one larva was successfully reared to full maturation and no TTX was detected.

The Inter-Valley Soil Comparative Survey: the ecology of Dry Valley edaphic microbial communities.

Recent applications of molecular genetics to edaphic microbial communities of the McMurdo Dry Valleys and elsewhere have rejected a long-held belief that Antarctic soils contain extremely limited microbial diversity. The Inter-Valley Soil Comparative Survey aims to elucidate the factors shaping these unique microbial communities and their biogeography by integrating molecular genetic approaches with biogeochemical analyses. Although the microbial communities of Dry Valley soils may be complex, there is little doubt that the ecosystem's food web is relatively simple, and evidence suggests that physicochemical conditions may have the dominant role in shaping microbial communities. To examine this hypothesis, bacterial communities from representative soil samples collected in four geographically disparate Dry Valleys were analyzed using molecular genetic tools, including pyrosequencing of 16S rRNA gene PCR amplicons. Results show that the four communities are structurally and phylogenetically distinct, and possess significantly different levels of diversity. Strikingly, only 2 of 214 phylotypes were found in all four valleys, challenging a widespread assumption that the microbiota of the Dry Valleys is composed of a few cosmopolitan species. Analysis of soil geochemical properties indicated that salt content, alongside altitude and Cu(2+), was significantly correlated with differences in microbial communities. Our results indicate that the microbial ecology of Dry Valley soils is highly localized and that physicochemical factors potentially have major roles in shaping the microbiology of ice-free areas of Antarctica. These findings hint at links between Dry Valley glacial geomorphology and microbial ecology, and raise previously unrecognized issues related to environmental management of this unique ecosystem.