The Foregut of Gecarcinus lateralis as an Organ of Salt and Water Balance

The permeability of the foregut of the land crab, Gecarcinuslateralis, to tritiated water (THO), Na²², and Cl³⁶ was studiedin vitro during the intermolt period and after ecdysis. In crabswith eyestalks, the foregut is permeable to water and ions inthe direction hemolymph-to-lumen and lumen-to-hemolymph, bothduring the intermolt period and after ecdysis. However, theforegut of animals without eyestalks is impermeable after ecdysis. The movement of THO always follows the movement of Na²² acrossthe wall of the foregut, while the movement of Cl³⁶ may or maynot be correlated with the movement of Na²² and THO. Comparisonof the ratio of water to ions in the hemolymph with the ratiocalculated from radioisotope flux indicates that Na⁺ and waterare probably moving isosmotically, although not necessarilyaccompanied by Cl^– When an extract of the thoracic ganglionic mass of G. lateralisis added to the "hemolymph side" of the foregut in vitro, thereis immediately a large increase in permeability to water andsalts. This occurs in the foregut of crabs with eyestalks duringintermolt and also in eyestalkless crabs after ecdysis. Thus, not only is the foregut of Gecarcinus lateralis permeableto water and salts in both directions, but also the extent ofits permeability is under neuroendocrine control. As a consequence,the animal may be able to move water and salts into the foregutor out of it into the hemolymph as needed. This may be an importantadaptation for a terrestrial crab that must conserve water,especially at the critical time of ecdysis.     

COMPETITIVE ENZYME IMMUNOASSAY FOR THE RAPID IDENTIFICATION OF SALMONELLA

A competitive enzyme immunoassay using a murine monoclonal antibody M105 directed against a genus-specific epitope in the Salmonella lipopolysaccharide was used to identify over 200 strains of Salmonella submitted to the National Laboratory for Enteric Pathogens. The immunoassay rapidly identified 208 strains of Salmonella representative of subspecies I, II, III_a, III_b, IV, and V, including 89 different serotypes from 26 O serogroups. The competitive enzyme immunoassay did not give positive results with 3 strains of Citrobacter freundii and 4 strains of Escherichia coli which were submitted to our laboratory as suspect Salmonella.     

Selection of a suitable cladoceran species for toxicity testing in turbid waters

Abstract Two Australian cladocerans, Moina australiensis Sars and a species of Ceriodaphnia, were evaluated as possible biological indicator organisms to assess the toxicity of irrigation supply and drainage water of the Murrumbidgee and Coleambally Irrigation Areas. M. australiensis, being large (～2000 μm) and orange, was initially chosen to overcome visibility problems in highly turbid Australian inland waters. However, the organism responded erratically in culture. Mortality was high and neonate production was unpredictable when cultured under recommended United States Environmental Protection Agency protocols. Attempts to improve culture performance by optimizing food (quality and quantity), water (control source, hardness, volume) and temperature were only marginally successful. Similar difficulties were not evident when Ceriodaphnia sp. was used as the test organism. Although Ceriodaphnia sp. is small (～1000 μm), grey and more difficult to see in turbid water its responses were more predictable and reliable than those of M. australiensis. Results of initial trials comparing the two organisms suggest that Ceriodaphnia sp. was a better test organism and more suited to local requirements.     

A Soluble Phospholipase of Toxoplasma gondii Associated with Host Cell Penetration

We previously reported that phospholipase increases host cell penetration by Toxoplasma gondii . Here we show that calcium-dependent phospholipase A (PLA) activity is found in the supernatant of sonically disrupted T. gondii . When fractions of disrupted T. gondii were incubated with host cells, the release of fatty acids and lysolipids was detected. Fractions of sonically disrupted T. gondii with PLA activity increased T. gondii host cell penetration in a bioassay. In addition, a protein of approximately 20 kDa was detected by immunoblot of T. gondii antigens with horse antiserum to snake venom, the major antibody of which recognizes PLA₂. Incubation of T. gondii with exogenous PLA₂ resulted in increased solubility of a rhoptry protein. This protein, which we previously characterized as involved with enhanced parasite invasion of host cells and which is recognized by monoclonal antibody Tg49, was detected in increased amounts in supernatant fractions of extracellular parasites treated with PLA₂. Whereas without PLA₂ treatment, it is only slightly soluble under physiological conditions. This raises the possibility that PLA may be implicated in the release of rhoptry proteins.     

The Nucleariid Amoebae: More Protists at the Animal-Fungal Boundary

Nucleariid amoebae are naked amoebae, generally characterized by a spherical or sometimes flattened body with radiating filopodia. Most species preferentially consume algal prey or cyanobacteria. Phylogenetic analyses of the small-subunit rRNA coding regions from four nucleariid amoebae place these species near the origin of the animal-fungal divergence, together with the choanoflagellate-Corallochytrium and the ichthyosporean clades. The species Nuclearia delicatula, N. moebiusi, and N. simplex form a monophyletic group, while ATCC 30864, tentatively but possibly incorrectly assigned to Nuclearia sp., represents a separate line of descent. These nucleariids are unrelated to the lineage containing the testate filose amoebae (Testaceafilosia). Our findings expand the morphological and phylogenetic diversity of protists at the animal-fungal divergence.     

APPLICATIONS OF TIME-RESOLVED FLUOROIMMUNOASSAY TO DETECT MAGNETIC BEAD CAPTURED ESCHERICHIA COLI O157:H7

A time-resolved fluorescence technique was developed to detect Escherichia coli O157:H7 in ground beef burger. After a 4.5 h enrichment period, streptavidin coated magnetic beads conjugated with biotin-labeled anti E. coli O157:H7 were used to capture the bacteria. The bacteria were, at the same time, also labeled by a nonfluorescent, europium (Eu)-tagged anti-E. coli O157:H7 antibody. The sandwiched bacterial complexes were then concentrated using a magnetic particle concentrator and washed to remove other solution components. Upon addition of an enhancement buffer, the Eu-labels were then released from the antibodies and chelated to nitrilo-triacetic acid (NTA) and trioctylphosphine oxide (TOPO) to form highly fluorescent Eu-(2-NTA)₃(TOPO)_2–3 micellar complexes. Delayed fluorescence associated with these complexes was measured and its intensity was used to estimate the original bacterial concentration spiked in hamburger. This approach was applied to detect E. coli O157:H7 spiked in hamburgers. The results indicated this method is able to detect 1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37C. Specificity studies indicated that the approach exhibited no or limited cross reactivity to Salmonella typhimurium, E. coli K-12 or Shigella dysenteriae spiked in hamburgers. Thus, the developed approach may be used as a rapid screening procedure for E. coli O157 bacteria in foods.     

Characterization of Pneumocystis carinii Preparations Developed for Lipid Analysis

Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non-P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10⁸-10⁹ organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80–95%.     

Recovery of three arctic stream reaches from experimental nutrient enrichment

1. Nutrient enrichment and resulting eutrophication is a widespread anthropogenic influence on freshwater ecosystems, but recovery from nutrient enrichment is poorly understood, especially in stream environments. We examined multi‐year patterns in community recovery from experimental low‐concentration nutrient enrichment (N + P or P only) in three reaches of two Arctic tundra streams (Kuparuk River and Oksrukuyik Creek) on the North Slope of Alaska (U.S.A.). 2. Rates of recovery varied among community components and depended on duration of enrichment (2–13 consecutive growing seasons). Biomass of epilithic algae returned to reference levels rapidly (within 2 years), regardless of nutrients added or enrichment duration. Aquatic bryophyte cover, which increased greatly in the Kuparuk River only after long‐term enrichment (8 years), took 8 years of recovery to approach reference levels, after storms had scoured most remnant moss in the recovering reach. 3. Multi‐year persistence of bryophytes in the Kuparuk River appeared to prevent recovery of insect populations that had either been positively (e.g. the mayfly Ephemerella, most chironomid midge taxa) or negatively (e.g. the tube‐building chironomid Orthocladius rivulorum) affected by this shift in dominant primary producer. These lags in recovery (of >3 years) were probably driven by the persistent effect of bryophytes on physical benthic habitat. 4. Summer growth rates of Arctic grayling (both adults and young‐of‐year) in Oksrukuyik Creek (fertilised for 6 years with no bryophyte colonisation), which were consistently increased by nutrient addition, returned to reference rates within 1–2 years. 5. Rates of recovery of these virtually pristine Arctic stream ecosystems from low‐level nutrient enrichment appeared to be controlled largely by duration of enrichment, mediated through physical habitat shifts caused by eventual bryophyte colonisation, and subsequent physical disturbance that removed bryophytes. Nutrient enrichment of oligotrophic Arctic stream ecosystems caused by climate change or local anthropogenic activity may have dramatic and persistent consequences if it results in the colonisation of long‐lived primary producers that alter physical habitat.     

The sensitivity of phytoplankton in Loch Leven (U.K.) to changes in nutrient load and water temperature

1. Loch Leven is a shallow, eutrophic lake in Scotland, U.K. It has experienced much change over the 30 years that it has been studied; this has primarily been due to reduced nutrient loads to the lake through active catchment management. Its recovery has been slow and, therefore, we used a phytoplankton community model (PROTECH) to test its sensitivity to changing nutrient loads and water temperature.
2. PROTECH was initialized to simulate the observed phytoplankton community in 1995 and was then repeatedly run through a combination of step-wise changes in water temperature and nutrient load (two treatments were simulated for nutrient load: one changing both nitrate and phosphorus, and one changing just phosphorus). The effect on total chlorophyll- a concentration, cyanobacteria abundance and phytoplankton diversity was examined.
3. Whilst changes in temperature had little effect, variations in the nutrient load produced a range of responses. Increasing only the phosphorus load caused a large increase in Anabaena abundance and total chlorophyll- a concentration. However, the opposite response was recorded when nitrate load was changed as well, with Anabaena increasing its biomass under reduced nutrient load scenarios.
4. The key factor determining the type of response appeared to be nitrogen availability. Anabaena , a nitrogen fixer, could exploit the phosphorus resource of Loch Leven under limiting nitrogen conditions, allowing it to dominate under most of the scenarios tested apart from those supplying extra nitrogen to the lake. The model predictions agree with the observed data, which show that Anabaena continues to dominate the summer phytoplankton bloom in Loch Leven despite the considerable reduction in phosphorus supply from the catchment. This research provides a possible explanation for this.     

Process‐Based Species Action Plans: an approach to conserve contemporary evolutionary processes that sustain diversity in taxonomically complex groups

Many endemic plant species belong to taxonomically complex groups. These endemics have often arisen as a consequence of recent and rapid evolutionary divergence facilitated by processes such as hybridization, polyploidy and/or breeding system transitions. The rapid and dynamic nature of divergence in taxonomically complex groups leads to problems in the implementation of traditional species‐based approaches for the conservation of the biodiversity that they contain. Firstly, the taxa of interest can be difficult to define and identify, leading to practical difficulties in implementing conservation measures. Secondly, a species‐based approach often fails to capture the complexity of diversity present in the taxonomically complex group. To accommodate these challenges, we have developed a Process‐Based Species Action Plan approach. This is designed to conserve the processes leading to the generation of biodiversity, rather than focusing on the preservation of individual named taxa. We illustrate the approach using a group of endemic tree species (Sorbus) on the Scottish island of Arran that have originated via a combination of multiple recent hybridization events and apomixis. The plan focuses on the optimization of habitat management to ensure the reproduction and regeneration of Sorbus in the zone in which these evolutionary processes operate, and to facilitate hybridization that will ensure the continued generation of diversity in this group. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 194–203.