Induction of Antibiotic Resistance in Paramecium tetraurelia by the Bacterial Gene APH-3'-II

We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neo^r) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neo^r ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10–20 picoliters of linearized PXV-NEO at > 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neo^r-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neo^r-specific DNA and neo^r-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.     

Influence of phytoplankton on the response of bacterioplankton growth to nutrient enrichment

1. Laboratory experiments were conducted to test the effect of nutrient enrichment on bacterioplankton growth in the presence and absence of phytoplankton. 2. In one series of experiments, bacterioplankton growth in terms of specific activity [³H-thymidine incorporation (cell number)^?1] was greater in whole lake water samples than in samples from which phytoplankton had been removed by filtration (1.0 μm), regardless of the nutrient enrichments (control, NH⁺₄ plus PO^3-₄ and mannitol). Organic C enhanced bacterioplankton growth in both whole and filtered lake water. 3. In another series of experiments (with the same nutrient enrichments as in the first experiment except that glucose replaced mannitol), bacterioplankton growth in whole lake water enriched with PO^3-₄ plus NH⁺₄ and incubated in the light was greater than in two treatments designed to inhibit photosynthetic activity (+DCMU and dark). Bacterioplankton response to nutrient addition was greatest in the PO^3-₄ plus NH⁺₄ enrichment under all three conditions (light +DCMU, and dark). 4. These results indicate that bacterioplankton growth could be directly limited by inorganic P and N when these elements are in short supply. Enhancement of bacterioplankton growth by phytoplankton occurs only under PO^3-₄ and NH⁺₄ replete environments.     

The effect of a foliar disease (rust) on the development of Gastrophysa viridula (Coleoptera: Chrysomelidae)

Abstract.

• 1 Gastrophysa viridula Degeer (Coleoptera: Chrysomelidae) and the pathogenic rust fungus Uromyces rumicis (Schum.) Wint. both occur on leaves of Rumex crispus L. and R.obtusifolius L. Individual stages of beetle development, and egg laying, were compared on healthy and infected leaves of each plant species in the laboratory. Oviposition choice was investigated in the field and laboratory.
• 2 Beetles reared on infected leaves of each species had greater larval mortality and slower development than those reared on healthy leaves. Although larvae feeding on infected leaves consumed up to 2.5 times more dry weight than those reared on healthy leaves, they had a lower relative growth rate and pupated at a lower weight. These changes were consistent with the reduced nutritive quality of rust-infected Rumex leaves.
• 3 Fecundity of beetles reared on infected leaves of both species was considerably reduced. Eggs laid by beetles feeding on infected R.crispus leaves also had a reduced viability.
• 4 The beetle developed consistently poorer on healthy R.crispus than on healthy R.obtusifolius throughout its life-cycle. Differences in larval performance were greater between host species than between infected and healthy leaves.
• 5 Oviposition was similar on infected and healthy R.crispus in both the laboratory and field. However, adults consumed less, and laid fewer eggs on infected than on healthy R.obtusifolius. The pattern of egg laying on different aged leaves was affected by rust infection: a greater proportion of eggs was laid on the older, infected leaves, than on the equivalent aged leaves on the healthy plants. Few larvae survived from eggs laid on rusted leaves in the field.

     

The effect of suspended clay on ciliate population growth rates

• 1 Renewed batch cultures of three algivorous and one bacterivorous ciliate showed that ciliate reproductive rates increase with increasing food concentration up to an asymptote. Maximum r_m values for Strobilidium gyrans, Bursaridium difficile and Euplotes eurystomus feeding on the cryptomonad alga Rhodomonas minuta were 0.87, 0.86 and 0.45 day-¹, respectively, while Paramecium aurelia feeding on the bacterium Enterobactor aerogenes had an r_m of 0.40 day-¹. B. difficile had a food threshold (r_m= 0) of 0.38 μg Cml-¹ and P. aurelia had a threshold of 1.6μg Cml-¹.
• 2 The presence of suspended fine (particle size <1 μm) and coarse (particle size 1–2μm) clay suppressed the half maximum reproductive rate of the ciliate Strobilidium gyrans by up to 69% but had no effect on B. difficile, E. eurystomus or P. aurelia. Populations of ciliates unaffected by clay may be released in situations where suspended particles negatively affect their metazooplankton predators and competitors.

     

Identification of predominant environmental factors structuring stream macroinvertebrate communities within a large agricultural catchment

• 1 Patterns of macroinvertebrate community composition were examined in streams within a 40000-km² catchment in central Michigan, U.S.A., to identify the major environmental gradients influencing community variation. Agriculture and associated clay and sandy soils predominated in much of the region.
• 2 Eighty macroinvertebrate taxa were collected from stream surveys conducted during May and August 1990. Community composition varied primarily by the proportions of Plecoptera and Ephemeroptera. Benthic communities from the heaviest agricultural zones were most different from those at other sites.
• 3 Chemical composition among the sites varied most in relation to nutrients (NH₃, NO3, PO₄). Other parameters were relatively similar. Physical characteristics of the sites were scored in six habitat categories: (i) substrate characteristics, (ii) instream cover, (iii) channel morphology, (iv) riparian zone and stream-bank conditions, (v) riffle/run quality, (vi) pool quality. Most physical habitat scores were lowest in the intense agriculture zones.
• 4 The relative importance of physical and chemical variables in explaining variation in macroinvertebrate communities was quantified using canonical correspondence analysis. Substrate characteristics were most important in both surveys. Significant correlations (P<0.05 and P<0.10) were observed between substrate quality and total numbers of Ephemeropteran, Plecopteran, and Trichopteran taxa. These relationships reflected correlations from sites in the clay soil-type region (P<0.01 and P<0.10) which contrasted with non-significant results from the less impacted, sandy soil-type region.
• 5 Effective stream restoration efforts in this region will require the alteration of local land-use activities that influence the physical habitat. Further development of empirical relationships between catchment activities and substrate and channel characteristics within natural geomorphic regions is essential for the evaluation of restoration projects.

     

Reproduction in two submersed macrophytes declines progressively at low pH

• 1 Greenhouse experiments tested the effect of pH 5 v pH 7.5 on reproductive success for the freshwater macrophytes Najas flexilis, an annual, and Vallisneria americana, which perennates as a tuber.
• 2 Seed production by the small Najas plants grown at pH 5 averaged 0.25 seeds/plant, in contrast to 95.5 seeds/plant at pH 7.5. At low pH, Vallisneria grown from seeds produced no flowers and too few tubers to replace themselves, so that sexual reproduction failed nearly completely for both species.
• 3 Vallisneria grown from tubers produced 97% less total tuber mass at pH 5 (0.4 v 14.9 g), the compounded result of producing, on average, 89% fewer and 82% smaller tubers. The smaller tubers developed at pH 5 were less likely to overwinter in the field, and those surviving tubers subsequently grew into smaller plants.
• 4 These findings generated the hypothesis of a closing spiral: growth at low pH (and relatively low CO₂ concentrations) results in small plants that produce a smaller number of progressively smaller tubers each autumn, which in turn develop into progressively smaller plants each summer. This hypothesis was supported by field transplant experiments in two acidic Adirondack Mountain (NY) lakes.

     

A simple periphyton sampler for algal biomass estimates in streams

• 1 A simple periphyton sampler, which is cheap and easy to construct, is described. The key component is a disposable scouring disc which also serves to adsorb detached material.
• 2 Field trials show that the scouring sampler is significantly more efficient at removing and retaining periphyton from stones in streams for biomass estimation than other commonly used techniques.
• 3 The scouring sampler requires only one operator. Its small size and speed of use facilitate replication of samples. Samples can be collected from stones as small as 30mm diameter.

     

Effects of Ultraviolet Irradiation on the Cell Cycle

Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20–30 mJ/cm² for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid-1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M “injury responses” of other cell types might be expected, consistent with their differentiated phenotypes.     

Aminopeptidases from Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei

ABSTRACT. Using fluorogenic substrates and polyacrylamide gels we detected in cell-free extracts of Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei only a single aminopeptidase. A comparative study of the aminopeptidase activity in each extract revealed that the enzymes have similar specificities and kinetics, a near-neutral pH optima of 7.2 and are moderately thermophilic. Each has an apparent molecular weight of 80,000 ± 10,000, determined by high performance liquid chromatography on a calibrated SW500 column. Whilst the P. c. chabaudi and P. berghei activity co-migrate in native polyacrylamide gels, that of P. falciparum migrates more slowly. The three enzymes can be selectively inhibited by ortho -phenanthroline and are thus metallo-aminopeptidases; however, in contrast to other aminopeptidases the metal co-factor does not appear to be Zn²⁺.     

New Species of Lagenophrys (Ciliophora,Peritrichia) from New Zealand and Australia1

Lagenophrys novazealandae n. sp. occurs on the gills of Paranephrops zealandicus, a parastacid crayfish from New Zealand. The new species has the hemispheroidal lorica most common among members of its genus and is distinguished by its possession of large tubercles on the thickened edge of the anterior lip of the lorica aperture, a deep cleft in the left side of the lip's edge, and a ovoid to reniform macronucleus located in the right-hand part of the body. It is probable that an as yet unnamed species of Lagenophrys known to occur on another species of Paranephrops in New Zealand is distinct from L. novazealandae but phylogenetically related to it. Lagenophrys petila n. sp. occurs on setae of Parastacoides tasmanicus, a parastacid from Tasmania. The new species has an ovoid lorica tapering to a slender pseudostalk at the posterior end, a type of lorica possessed by only two other members of its genus that also attach to their host's setae. It is distinguished from the other ovoid species by the proportions of the lorica, the extreme shortness of the lips of the lorica aperture, and an ovoid macronucleus located in the right, anterior part of the body. Clefts in the lips of L. novazealandae and other members of Lagenophrys may function as points of flexure to allow the lips to bend in ways that accommodate interspecific differences in the size of the epistomial disk and its operation during suspension feeding.