Chemorepellents in Paramecium and Tetrahymena

Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. We have used a convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t-maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels.     

HUNGER OVERCOMES FEAR IN NASSARIUS FESTIVUS, A SCAVENGING GASTROPOD ON HONG KONG SHORES

On protected intertidal sand flats, virtually worldwide, nassariidsnails scavenge carrion. Typically, they lie in repose beneaththe sand surface but emerge to feed when chemoreceptors detectcarrion. Distance from the potential food influences whetherthey move towards it and, if they do, whether they obtain ameal before it is consumed by others. This study shows that the rate at which Nassarius festivus movestoward food bait is similar for starved and well-fed individuals.That is, when individuals decide to feed, the rate at whichthey move toward food is always approximately the same. Thisstudy exposes another facet of nassariid nutrition related tothe degree of hunger. Recently fed individuals, i.e., thosewhich have fed for < 14 days, will cease feeding and departpalatable food if crushed conspecifics are added to it. Individualswhich have not fed for > 14 days tend to remain feeding,despite the addition of crushed conspecifics. Another studyhas shown that N. festivus consumes sufficient energy from afed-to-satiation meal to sustain it in repose for 20 days.These experiments clarify the observation. Between 14 to 20days since its last meal, N. festivus will feed when the possibilityarises, despite potentially adverse consequences. That is, hungerovercomes the fear of being consumed at a feeding assemblagebecause, without feeding, the alternative may be death by starvation.Thus, a nassariid makes decisions about whether or not to feed.The fear of predation causes N. festivus to abandon a meal ifthe previous one was obtained within a fortnight but, thereafter,it will risk predation to avoid starvation. (Received 14 March 1994; accepted 7 July 1994)     

Effective Elimination of Fibroblasts in Cultures of Melanocytes by Lowering Calcium Concentration in TPA Depleted Medium Following Geneticin Treatment

Melanocytes grow well in culture media supplemented with phorbol ester. However, phorbol ester is metabolically stable and has prolonged effects on multiple cellular responses. Different groups have reported that they could cultivate melanocytes without phorbol ester. However, in medium without phorbol esters contaminating fibroblasts are often difficult to eliminate. We have developed an improved method for eliminating fibroblasts using geneticin in serum free medium with bFGF, endothelin-1 and no phorbol ester. Treatment with a low concentration of geneticin effectively eliminated contaminating fibroblasts and was not harmful to melanocytes if geneticin was added initially in a high calcium media (2 mM) followed by a change to a low (0.09 mM) calcium media immediately after removal of the geneticin. With this technique we could easily establish pure melanocyte culture. This culture method will provide a useful tool for studies on melanocyte cellular response without the influence of phorbol ester.     

Long-Term Storage of Infective Microsporidian Spores in Liquid Nitrogen

Thirty-one species of microsporidia, isolated from insects and stored in liquid nitrogen for up to 25 yr, were infectious when removed from liquid nitrogen. The natural hosts of all of these microsporidia were terrestrial insects, representing six different insect orders: Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, and Orthoptera. All microsporidia from terrestrial insects that were tested survived storage in liquid nitrogen, while Nosema algerae , a microsporidium from aquatic mosquito hosts did not survive freezing in liquid nitrogen. A Nosema species from the alfalfa weevil, Hypera postica , lost some infectivity in a water storage medium after 25 yr in liquid nitrogen. Liquid nitrogen storage of microsporidian spores in 50% and 100% glycerol media reduced loss of infectivity and is recommended for extended storage of microsporidia from terrestrial insect hosts.     

Osmotic and Metabolic-Induced Changes in Light Scattering of Leishmania donovani as Measured by Flow Cytometry

Leishmania donovani promastigotes were collected from cultures in log and stationary phases of growth and resuspended in Hank's Balanced Salt Solution containing 1 mM sodium acetate. Changes in the forward and side scattering of the cells were measured by flow cytometry in response to acute changes in osmolality and to the addition of several different substrates. Forward and side scattering of cells from log phase cultures decreased when the osmolality was decreased by the addition of H₂O and increased when the osmolality was increased by the addition of NaCl. Cells from stationary phase cultures had about the same forward scatter as cells from log phase cultures, but almost a four-fold lower side scatter, and their side scatter values did not change significantly in response to a reduction in osmolality. Microscopic observation showed that both log and stationary cells got longer and thinner, on average, in response to hyperosmolality. The light scattering properties of log (but not of stationary) cells changed in a reproducible manner when substrates were added to the buffer. The ratio of forward to side scatter increased in the following order: controls in balanced salt solution >aspartate >glutamate, glucose or 2-deoxyglucose >alanine >proline. Thus the light scattering properties of L. donovani promastigotes change with culture age, in response to changes in osmolality, and, in log phase cells, in response to the presence of several substrates.     

Identification of CO2 Chemoreceptors in Helix pomatia

SYNOPSIS. Gas exchange in pulmonate snails of the family Helicidaeoccurs through a highly vascularized diffusion lung known asthe mantle. The extent of ventilation of the mantle dependsupon the duration and size of opening of an occlusible poreknown as the pneumostome. In Helix aspersa and Helix pomatia,pneumostomal size and frequency of opening are exquisitely sensitiveto CO₂. Respiratory CO₂ chemosensitivity resides in a discreteregion of the subesophageal ganglia. The discharge pattern ofmany neurons in the chemoreceptor area changes during stimulationwith CO₂. However, the electrophysiological response to CO,stimulation alone does not discriminate between CO₂ chemoreceptorcells and CO₂-insensitive neurons active in the pneumostomalresponse to CO₂. We identified a subset of CO₂-sensitive neuronsfrom the larger population of neurons active during CO₂ stimulation.The action potential discharge frequency of CO₂ chemosensoryneurons increased in response to CO₂ stimulation. An increaseddischarge frequency of CO₂-sensitive neurons was associatedwith increased pneumostomal opening, and both the size and thefrequency of pneumostomal opening increased during CO₂ stimulation.Injecting depolarizing current into individual chemosensoryneurons elicited opening of the pneumostome in the absence ofCO₂. Action potential generation in response to CO₂ was independentof synaptic transmission. Removal of individual CO₂-sensitivecells or inhibition of action potential generation in CO₂-sensitivecells reduced or eliminated pneumostomal responses to CO₂. CO₂sensitivity in chemoreceptor cells required extracellular calcium,but not sodium. Substituting barium for calcium supported chemoreceptoractivity. In summary, we have identified respiratory related,chemosensory neurons that are CO₂ sensitive in the absence ofsynaptic input.     

Phylogenetic relationships among marsupial frog genera (Anura: Hylidae: Hemiphractinae) based on evidence from morphology and natural history

We reassess the phylogenetic relationships of genera of hemiphractine hylid frogs (Marsupial Treefrogs) and discuss the evolution of several distinctive characters within this group using parsimony analysis. Fifty-one morphological and life-history characters were sampled from two species of Cryptobatrachus , three species of Flectonotus , 17 species of Gastrotheca , all five species of Hemiphractus , and one species of Stefania as the ingroup and three hyline, one phyllomedusine, and one pelodryadine species as outgroups. Our results support the mon-ophyly of Flectonotus, Cryptobatrachus , and Hemiphractus. Gastrotheca is paraphyletic with respect to Hemiphractus , dorsal pouches were lost in the ancestor of Hemiphractus. Direct development is a synapomorphy for Hemiphractinae and tadpoles were regained independently several times. These results stand in stark contrast to the prevailing paradigm regarding marsupial frog relationships.     

Indirect effects of metal contamination on energetics of yellow perch (Perca flavescens) resulting from food web simplification

1. Benthic invertebrate community composition and yellow perch (Perca flavescens) diet, growth and activity levels from lakes along a metal‐contamination gradient were used to assess the importance of a naturally diverse prey base for maintaining energy transfer to growing fish, and how this transfer is disrupted by metal contamination. 2. Zoobenthic communities had lower diversity in metal‐contaminated lakes, with a notable absence of large bodied invertebrate taxa. 3. The average mass of non‐zooplankton prey items was significantly greater for 2+ and 3+ perch from the reference lake, and increased significantly with age in all except the most contaminated lakes where prey choice was limited. 4. Benthivorous perch from all contaminated lakes exhibited slowed growth. Perch from one of the contaminated lakes exhibited faster growth during piscivory, indicating slowed growth only while benthivorous. 5. Estimates of fish activity, using the activity of the glycolytic enzyme Lactate dehydrogenase in perch white muscle tissue as a proxy, suggested that shifts in diet to larger prey (in reference and intermediately contaminated lakes) lowered activity costs, which may explain how diet shifts maintain growth efficiency as perch grow larger.     

Ultrastructural Observations on the Infection of Rat Liver by Plasmodium berghei Sporozoites In Vivo1

The invasion of liver parenchymal cells by sporozoites of Plasmodium berghei Vincke & Lips, 1948, was studied in vivo using transmission electron microscopy. Livers of Brown Norway rats were examined 30 and 60 min after intraportal injection of 15 million sporozoites each. Sporozoites found after incorporation into vacuoles in hepatocytes were often located near a bile canaliculus at the lateral cell surface, surrounded by hepatocyte lysosomal structures; however, degradation of sporozoites caused by lysosomal digestion inside hepatocytes was never observed. Due to the crescent shape of sporozoites, serial sections were necessary to demonstrate the actual process of invasion of the hepatocyte. The hepatocyte's plasmalemma appeared to invaginate due to the sporozoite's action, thereby creating a parasitophorous vacuole. It was suggested that the sporozoite actively penetrated the hepatocyte; however, no visible depletion of rhoptries and micronemes was observed.