Acid Intracellular Vesicles and the Cytolysis of Mammalian Target Cells by Entamoeba histolytica Trophozoites1

ABSTRACT. Entamoeba histolytica kills mammalian target cells in a multi-step sequential process with separate adherence, cytolytic, and phagocytic events. In the studies reported here, we used fluorescein isothiocyanate linked to dextran to label the endocytic vesicles of the HM1 strain of E. histolytica and measure vesicle pH (5.1 ± 0.2 by spectrofluorimetry). Concentrations of NH₄Cl (1.0–10.0 mM) sufficient to increase vesicle pH to °5.7 inhibited amebic killing of target Chinese hamster ovary (CHO) cells as assayed by trypan blue staining, by the release of ³H-thymidine previously incorporated into CHO cell monolayers, and by the release of ¹¹¹indium oxine from radiolabeled CHO cells. Similar effects were also observed with two other weak bases, primaquine and chloroquine (both 50 μM). In contrast, NH₄Cl (10 mM) did not affect either the adherence or phagocytic events, as measured by amebic adherence to CHO cells at 4°C and by the binding and ingestion of ³H-leucine-labeled bacteria. In the presence of NH₄Cl and and the carbohydrate ligand asialofetuin, there was no evidence of intracellular trapping of the amebic galactose-inhibitable lectin; inhibition of adherence by cycloheximide (10 μg/ml for 3 h) suggested rapid turnover of the surface lectin. Prolonged exposure to NH₄Cl for 48 h (which had no effect on amebic protein synthesis) or shorter exposure to cycloheximide (10 μg for 3 h) produced persistent inhibition of cytolysis. These results indicate that an uninterrupted acid pH in intracellular endocytic vesicles is necessary for the cytolysis of target cells by E. histolytica trophozoites.     

The Effect of the Quantitative Protargol Stain and Lugol's and Bouin's Fixatives On Cell Size: A More Accurate Estimate of Ciliate Species Biomass

ABSTRACT. The quantitative protargol stain (QPS) is used to estimate ciliate biomass and species composition from mixed field samples. Length, width, breadth and volume of live Euplotes sp., Eutintinnus sp., Strobilidium spiralis, Strombidium acutum , and Gymnodinium sanguineum were compared with 0.6% acid Lugol's fixed, 5% Bouin's fixed, and QPS cells. Cells shrank due to treatments (ANOVA and Tukey's test, α= 0.05). Protistan post-fixation cell volume (as a percentage of live volume) was 55%-80% for acid Lugol's fixed, 40%-70% for Bouin's fixed, and 30%-65% for QPS. Each species shrank to a different extent; cytostructural elements apparently alter the effect of fixation. Egestion is likely not the main cause of shrinkage since the autotroph, G. sanguineum , shrank to the same extent as the heterotrophs when stained by QPS. If field studies do not consider fixation effects on cell size, biomass may be underestimated. We recommend, for studies on planktonic ciliates, either acid Lugol's and QPS be used concurrently or QPS be used alone and biovolume values divided by 0.4 to correct for shrinkage. We stress that this is a rough estimate as this value ranges from 0.3 to 0.45 for planktonic protists.     

The Cellular Regulation of Vesicle Exocytosis by Entamoeba histolytica1,2

ABSTRACT. We studied the cellular regulation of vesicle exocytosis by Entamoeba histolytica utilizing release of endocytosed ¹²⁵iodine (¹²⁵I) labeled tyrosine conjugated dextran; ¹²⁵I-dextran entered the acid pH vesicles of the amebae and was not degraded during these studies. Exocytosis was temperature dependent with 74%, 36%, 4%, and 0% of ¹²⁵I-dextran released after 120 min at 37°C, 31°C, 25°C, and 4°C, respectively (P < 0.01 for each). Exocytosis at 37°C was inhibited by cytochalasin D (10 μg/ml), EDTA (10 mM), or the putative intracellular calcium antagonist TMB-8 (250 μM) (P < 0.01 for each at ≥ 60 min). Calcium ionophore A23187 (1 μM) enhanced exocytosis at 5 and 15 min (P < 0.01). Elevation of vesicle pH with NH₄Cl (10 mM) had no effect on release of ¹²⁵I-dextran; phorbol myristate acetate (10^?6 M) increased exocytosis by 46% at 30 min (P < 0.01). Centrifugation of amebae with target Chinese hamster ovary cells resulted in decreased ¹²⁵I-dextran release into the cell supernatant after 30 and 60 min at 37°C (by 40% and 42%, respectively, P < 0.01); release of ¹²⁵I-dextran returned to control values with addition of 1.0 g% galactose or GalNac but not with mannose or N-acetyl-D-glucosamine. Amebic phagocytosis of serum-exposed latex beads had no effect on release of dextran by amebae (n = 16). Exocytosis of acid pH vesicles by E. histolytica is temperature-, microfilament-, and calcium-dependent, and stimulated by phorbol esters.     

Evolution on a volcanic conveyor belt: using phylogeographic reconstructions and K–Ar-based ages of the Hawaiian Islands to estimate molecular evolutionary rates

The Hawaiian Islands form as the Pacific Plate moves over a 'hot spot' in the earth's mantle where magma extrudes through the crust to build huge shield volcanos. The islands subside and erode as the plate carries them to the north-west, eventually to become coral atolls and seamounts. Thus islands are ordered linearly by age, with the oldest islands in the north-west (e.g. Kauai at 5.1 Ma) and the youngest in the south-east (e.g. Hawaii at 0.43 Ma). K–Ar estimates of the date of an island's formation provide a maximum age for the taxa inhabiting the island. These ages can be used to calibrate rates of molecular change under the following assumptions: (i) K–Ar dates are accurate; (ii) tree topologies show that derivation of taxa parallels the timing of island formation; (iii) populations do not colonize long after island emergence; (iv) the coalescent point for sister taxa does not greatly predate the formation of the colonized younger island; (v) saturation effects and (vi) among-lineage rate variation are minimal or correctable; and (vii) unbiased standard errors of distances and regressions can be estimated from multiple pairwise comparisons. We use the approach to obtain overall corrected rate calibrations for: (i) part of the mitochondrial cytochrome b gene in Hawaiian drepanidines (0.016 sequence divergence/Myr); (ii) the Yp1 gene in Hawaiian Drosophila (0.019/Myr Kambysellis et al. 1995 ); and (iii) parts of the mitochondrial 12S and 16S rRNA and tRNA^val in Laupala crickets (0.024–0.102/Myr, Shaw 1996 ). We discuss the reliability of the estimates given the assumptions (i–vii) above and contrast the results with previous calibrations of Adh in Hawaiian Drosophila and chloroplast DNA in lobeliods.     

ATMOSPHERIC AND UNDERWATER PROPAGATION OF BULLFROG VOCALIZATIONS

ABSTRACT

Male bullfrogs vocalize while partially submerged in shallow freshwater ponds. This imposes two potential propagation pathways, atmospheric and underwater, on transmission of their communication sounds. Propagation of pure tones, amplitude modulated (AM) broadband noise and natural calls was measured in air and underwater at three bullfrog breeding sites. In air, propagation losses were consistent with spherical spreading. No excess attenuation was observed for any tone frequency at any site. Both temporal envelope modulations and spectral cues are available to conspecific receivers at biologically realistic distances. The bullfrog's advertisement call is thus well adapted for transmission in air at the air/water interface. Underwater signal propagation differed at the three sites, consistent with substrate effects. Tone propagation showed the high-pass frequency window characteristic of shallow water. Broadband signals underwent propagation losses greater than expected by cylindrical spreading. Modulations of the envelope of natural calls remained discernible at distances where frequency-dependent propagation losses distorted the shape of the spectrum. Measurements of the propagation of the advertisement call emitted by a chorusing frog at the air/water interface confirm that periodicity cues embedded in the envelope are available to receivers both in air and underwater. High frequency cues available underwater overlap the maximal hearing sensitivity of larval conspecifics (tadpoles).     

High floral resource density leads to neural constraint in the generalist,floriphilic katydid,Phaneroptera brevis (Orthoptera: Phaneropterinae)

1. The neural constraint hypothesis has been investigated in the field of insect–plant interactions. It predicts that the consumer's detection ability, time spent on resources, and efficiency in resource use are affected by different aspects of resource availability. 2. The applicability of this hypothesis in florivory, however, generally lacks a mechanistic understanding of the effect of resource density on neural constraint, so using caged experiments, the (i) ability of a generalist, floriphilic katydid [(Phaneroptera brevis (Serville)] to find a floral resource (inflorescences of Bidens pilosa L.), (ii) attentiveness of the katydid on this resource after successfully detecting the floral resource, and (iii) efficiency of resource use under varying densities of the floral resource were studied. 3. The present results indicated no evidence of an obvious effect of floral resource density on the detection of resources by the florivore but an increase in floral‐resource density generally led to lower efficiency and attentiveness in the foraging katydids. 4. These findings provide the first evidence of the neural constraint hypothesis in the context of resource density and corroborate the applicability of the neural constraint hypothesis in florivory.     

Dispersal and foraging behaviour of Platygaster californica: hosts can't run, but they can hide

Abstract.  1. Host–parasitoid models often identify foraging behaviour and dispersal distance as important for system persistence.
2. Laboratory observations and field trials were used to characterise foraging behaviour and dispersal capability of Platygaster californica Ashmead (Platygasteridae), a parasitoid of the gall midge Rhopalomyia californica Felt (Cecidomyiidae).
3. Although foraging parasitoids meticulously searched plants in laboratory observations, none of the laboratory trials resulted in 100% parasitism, and the proportion of parasitism declined as midge egg density increased.
4. The field trials showed that the distribution of parasitism over distance from a central release point was hump-shaped, as predicted by a simple diffusion model. Mean parasitoid dispersal distance was 4.5 m, considerably farther than the 1.7 m mean midge dispersal found in previous work.
5. Although the parasitoid appears to search thoroughly for midge eggs and to disperse farther than the midge, the results of this study show how this host–parasitoid system may persist due to spatially variable incomplete parasitism.