MEMBRANE LIFTING TECHNIQUE FOR RAPID DETECTION OF ESCHERICHIA COLI 0157:H7 IN INOCULATED GROUND MEATS

A membrane lifting technique was developed for direct rapid detection of Escherichia coli 0157:H7 in inoculated ground meats. Duplicate groups of 2 meatballs were inoculated with volumes of 0.1-ml of a serial dilution (1:10) of E. coli 0157:H7 or a mixed culture containing one strain of E. coli 0157:H7 and a non-0157:H7E. coli serotype (E. coli ATCC 25922). Each meatball was sampled by sandwiching with 2 pieces of nitrocellulose membranes and pressing against each other to the center of the meatball. The membranes were in contact with the meats for 10 min to lift the bacteria. The membranes were removed and incubated on MacConkey-sorbitol agar plates with the meat contact side up. After 18 h incubation at 37C, an immunostain was performed directly on the membranes for detection of the presence or absence of E. coli 0157:H7. This method was found to be sensitive enough to detect as few as 1 to 2 cells of E. coli 0157:H7 inoculated on surfaces of 18-g meatballs. This method might be used as a rapid presumptive test for E. coli 0157:H7.     

Primary Structure and Phylogenetic Relationships of Glyceraldehyde-3-Phosphate Dehydrogenase Genes of Free-Living and Parasitic Diplomonad Flagellates

ABSTRACT. Complete nucleotide sequences have been established for two genes (gap1 and gap2) coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) homologs in the diplomonad Giardia lamblia. In addition, almost complete sequences of the GAPDH open reading frames were obtained from PCR products for two free-living diplomonad species, Trepomonas agilis and Hexamita inflata, and a parasite of Atlantic salmon, an as yet unnamed species with morphological affinities to Spironucleus. Giardia lamblia gap 1 and the genes from the three other diplomonad species show high similarity to each other and to other glycolytic GAPDH genes. All amino-acyl residues known to be highly conserved in this enzyme are also conserved in these sequences. Giardia lamblia gap2 gene is more divergent and its putative translation reveals the presence of a cysteine and serine-rich insertion resembling a metal binding finger. This motif has not yet been noted in other GAPDH molecules. All sequences contain an S-loop signature with characteristics close to those of eukaryotes. In phylogenetic reconstructions based on the derived amino acid sequences with neighborjoining, parsimony and maximum-likelihood methods the four typical GAPDH sequences of diplomonads cluster into a single clade. Within this clade, G. lamblia gap1 shares a common ancestor with the rest of the genes. The latter are more closely related to each other, indicating an early separation of the lineage leading to the genus Giardia from the lineage encompassing the morphologically less differentiated genera, Trepomonas, Hexamita and that of the unnamed species. This result is discordant with the orthogonal evolution of diplomonads suggested on the basis of comparative morphology. In neighbor-joining reconstructions G. lamblia gap2 occupies a variable position, due to its great divergence. In parsimony and maximum likelihood analysis however, it shares a most recent common ancestor with the typical G. lamblia gap1 gene, suggesting that it diverged after the separation of the Giardia lineage. The position of the diplomonad clade in broader phylogenetic reconstructions is firmly within the typical cytosolic glycolytic representatives of GAPDH of eukaryotes.     

Biofilm development and invertebrate colonization of wood in four New Zealand streams of contrasting pH

1. Biofilm development and activity on wood substrata (Nothofagus menziesii) were examined at four forested sites in a South Island, New Zealand, river catchment over a period of 6 months. Two of the sites had brown waters and mean pH of 3.7 and 4.5, whereas the other two had clear waters and mean pH of 6.3 and 6.8. 2. Fungi and other filamentous heterotrophs were the dominant colonizers of wood at all sites; few algal cells were present. Incorporation of ¹⁴C-glucose by biofilms was greatest in all four streams after 3 months, whereas endocellulase activity fluctuated over time and temporal patterns differed among streams. 3. No clear relationship was found between the incorporation of ¹⁴C-glucose or endocellulase activity of biofilms and pH, although at one near-neutral pH site ¹⁴C-glucose uptake increased in response to nutrient (N + P) additions. 4. After 6 months, incorporation of ¹⁴C-glucose and endocellulase activity of biofilms on Pinus radiata dowels buried vertically in the stream beds did not differ at depths of 3–9 cm and 19–25 cm in each stream. 5. Radiotracer experiments with a grazing amphipod (Paraleptamphopus sp.) demonstrated that biofilms on wood from all four sites could be ingested and at least partially assimilated. Chironomid larvae and harpacricoid copepods were the most abundant invertebrates colonizing wood substrata at all sites. Different chironomid species dominated at acidic and near-neutral pH sites. 6. Overall, our findings provide little support for the hypothesis that microbial activity on organic substrata is necessarily lower in streams of low pH.     

The postcranial skeleton of the Devonian tetrapod Tulerpeton curtum Lebedev

Postcranial remains of the Russian Late Devonian tetrapod Tulerpeton include the hexadactylous fore limb, hind limb, anocleithral pectoral girdle, squamation, and associated disarticulated postcranial bones. A cladistic analysis indicates that Tulerpeton is a reptiliomorph stem-group amniote and the earliest known crown-group tetrapod: Acanthostega and Ichthyostega are successively more derived plesion stem-group tetrapods and do not consititute a monophyletic ichthyostegalian radiation. Previous analyses suggesting a profound split in tetrapod phylogeny are thereby corroborated, and likewise the interpretation of Westlothiana as a stem-group amniote. The divergence of reptiliomorphs from batrachomorphs occurred before the Devonian-Carboniferous boundary. Tulerpeton originates from an entirely aquatic environment with a diverse fish fauna. The morphologies of its limbs and those of Devonian stem-tetrapods suggest that dactyly predates the elaboration of the carpus and tarsus, and that Polydactyly persisted after the evolutionary divergence of the principal lineages of living tetrapods. The apparent absence of a branchial lamina and gill skeleton suggests that Tulerpeton was primarily air-breathing, whereas contemporary stem-group tetrapods and more recent batrachomorphs retained greater emphasis on gill-breathing.     

The encrustation of fossil and recent sea-urchin tests: ecological and taphonomic significance

A comparison of fossil ( Echitwlampas sp. from the Lower Miocene Zogelsdorf Formation, Austria) and Recent ( Schizaster canaliferus from the northern Adriatic Sea) irregular sea-urchin tests shows that, upon their death, burrowing echinoids can serve as a substrate for a dense epigrowth. Size, shape, stable orientation, and surface residence-time were identified as key factors governing encrustation. The encrusters on fossil Echinolampas were bryozoans, serpulid polychaetes, barnacles, and coralline algae, while the recent material was initially encrusted by serpulids and hydrozoan colonies, and ultimately covered by the full range of sessile, hemi-sessile, and vagile species characterizing the benthic community in the Adriatic. In Echinolampas , epigrowth was more abundant on the lower (oral) surface; this specific distribution was echoed in S. canaliferus , where epigrowth started on the bottom side and grew upward. This indicates that the tests have a stable orientation and a surface residence-time long enough to allow intense encrustation. A taphonomic model is developed, and the role of encrustation on such special substrates for overall community structure is discussed. The Recent/fossil comparison provides new insights for both fields of study: the recent material indicates the role of soft-bodied faunas as well as the complexity of small-scaled ecological processes; the fossil material reflects many of the above phenomena and adds important taphonomic details on the fate of encrusted biogenic structures and on encruster growth patterns and distributions.     

The effects of domestic compost upon the germination and emergence of barley and six arable weeds

In a bioassay, leachate from composted household waste was found to decrease both cress germination and the germination of barley and six arable weeds, especially in the light. The effect of compost depth upon the emergence of these species was examined in a glasshouse experiment. Compost had no significant effect on barley emergence. However, weed emergence, particularly of those with small seeds, was reduced greatly by the physical effect of compost and, to a lesser extent, by its chemical effect. The emergence of the large-seeded species was relatively unaffected by increasing depths of compost, whereas that of the smaller-seeded weeds steadily decreased, being almost completely prevented by a 3 cm layer. The use of compost to control arable weeds in the field is discussed.     

ECOLOGY AND EVOLUTION OF MATING SYSTEMS OF FIDDLER CRABS (GENUS UCA)

1. General accounts of the natural history and behaviour of fiddler crabs suggest there exist two broad mating patterns in the genus. Most western and Indo-Pacific species mate on the surface of intertidal substrates near burrows females defend. The sexes associate only briefly during courtship and mating. In contrast, males of many American species court from and defend burrows to which females come for mating. Copulation occurs underground in burrows plugged at the surface; the sexes usually remain together for at least several hours. Here we summarize and contrast recent detailed field studies of the mating systems of U. pugilator, an American species, and U. vocans, a species widely distributed in the western and Indo-Pacific. We indicate how differences in the breeding ecology of these two species may account for basic differences in modes of sexual selection leading to the two broad mating patterns in the genus. 2. U. pugilator burrows in protected sandy substrates in the upper intertidal and supratidal zone. During ebb tide, nonbreeding crabs leave burrows they occupy during high tide to forage on food-rich substrates in the lower intertidal zone. Reproductively active males remain in the burrow zone where they fight for and defend burrows from which they court. Large males win most fights for burrows and tend to defend burrows high on the elevation gradient, especially during periods with relatively high tides. Females usually approach and descend the burrows of several males before choosing their mates by remaining in males' burrows. Males remain underground with their mates for 1–3 days until after they oviposit their eggs. Some males then emerge and leave their burrows while others sequester their mates in the chambers where mating and oviposition has occurred, dig new chambers and resume courtship, perhaps attracting additional females. In either case, females remain underground for approximately 2 weeks, finally emerging to release their planktonic larvae. Burrows that do not collapse due to tidal inundation or flooding by groundwater are best for breeding and usually are located relatively high on the elevation gradient. Females choose mates indirectly by preferring to breed in burrows that will remain intact while they oviposit and incubate their eggs. Large males mate more often than small males because they are better able to defend burrows at locations females prefer to breed. The mating system of U. pugilator may be classified as resource-defence polygyny. 3. U. vocans burrows in open muddy substrates in the mid- to lower intertidal zone. At a site near Chunda Bay, Australia, where the reproductive behaviour of this species has been studied in depth, both sexes feed near burrows they defend. Females tend to occupy their burrows for longer periods and move shorter distances than do males. Mating occurs on the surface near the burrows that females defend. Females accept both resident and wandering males as mates. They show no preference for mating with larger males. Female choice may be based on other male morphological or behavioural characteristics. Females oviposit their eggs either while on the surface or in their burrows. They produce relatively small clutches and are active on the surface throughout their breeding periods. Males fight both their neighbours and wandering males. Large males tend to win fights and defend burrows in areas where large females, which produce relatively many eggs, are most dense. Such areas may offer greater protection from predators than areas occupied by smaller females. Small males mate about as often as large males but may father fewer larvae. The mating system of U. vocans is resource-free and promiscuous. 4. The mating systems of U. pugilator and U. vocans differ fundamentally in that female U. pugilator require access to a specific microenvironment to breed successfully, while female U. vocans do not. We suggest this difference occurs because of contrasts in clutch sizes and the mobility and movement patterns of feeding females. Female U. pugilator produce relatively large clutches and probably experience more intense selection from factors that can cause egg loss and mortality than do U. oocans, which produce clutches of sufficiently small volume to be protected by their abdominal flaps. Hence, the range of suitable breeding environments for U. pugilator is small compared to that for U. vocans. In addition, U. pugilator burrows in areas that are relatively food-poor, leading to daily migrations to and from food-rich substrates in the lower intertidal zone, preventing female defence of an area suitable for both breeding and feeding. U. vocans, however, burrows in areas sufficiently rich to support feeding, leading to relatively low female mobility and defence of burrows that are also suitable breeding sites. 5. Adaptive radiation of the genus Uca in the Americas is manifest by trends toward smaller adult size, higher population densities, more frequent microgeographic sympatry and increased terrestriality, compared to species in the western and Indo-Pacific regions. We outline the general features of the selection mechanisms tying each of these trends to the evolution of resource—defence mating systems. Intraspecific variation in the courtship behaviour and site of mating in U. lactea and U. vocans supports our contention that resourse—defence behaviour tends to occur at high population densities. Additional data are needed to evaluate the other hypotheses critically.     

Enzymology of Plasma Membranes of Insect Intestinal Cells

The enzymology of insect intestinal cell plasma membranes isa field of scientific research that is in the earliest stagesof development. In this paper the few published studies specificallydesigned to isolate plasma membranes from insect intestinalcells and determine the enzymes associated with them are reviewedin light of both older studies that approached these problemsless directly and recent results from our laboratory. In the past few years reliable methods have been developed forthe isolation of specific portions of plasma membranes fromthe epithelial cells of the midguts of a few insect larvae.These membrane preparations have been assayed for a varietyof enzyme activities. Alkaline phosphatase, leucine aminopeptidaseand -glutamyl transpeptidase have shown promise as potentialmarkers for the plasma membranes of insect larval midgut cells.However, only the latter enzyme currently stands unchallengedas a marker for the apical portion of the plasma membrane ofinsect midgut columnar epithelial cells. No enzymes can yetbe considered to be even tentatively established as markersfor the basal or lateral portions of insect intestinal cells.     

Seasonal changes of osmotic pressure, symplasmic water content and tissue elasticity in the blades of dune grasses growing in situ along the coast of Oregon

Abstract Midday water potentials of blades of the dune grasses Ammophila arenaria (L.) Link and Elymus mollis Trin. ex Spreng. growing in situ declined over the summer growing period, indicating a trend of increasing water stress. An analysis of the water relations characteristics of these blades using pressure-volume techniques demonstrated that both species increased bulk osmotic pressure at full hydration () and, therefore, bulk turgor as an acclimation response. In A. arenaria, however, the increase of osmotic pressure (+ 0.35 MPa) was entirely the result of decreasing symplasmic water content. The increase of osmotic pressure (+ 0.54 MPa) observed in E. mollis blades was due to solute accumulation (72% of Δ) and to a lesser degree, decreased symplasmic water content (28% of Δ). Osmotic adjustment in E. mollis blades was accompanied by a significant decrease in tissue elasticity (_max went from 12 to 19 MPa). The elastic properties of A. arenaria blades remained constant over the same period and had a maximum modulus (10 MPa) that was always less than that of E. mollis, As estimated from Höfler plots, these seasonal adjustments of osmotic pressure and differences in tissue elasticity enabled plants in situ to maintain turgor pressure in the range of 0.5–0.6 MPa at the lowest water potentials of mid-August. Laboratorygrown plants exhibited the species-specific differences in osmotic pressure, turgor pressure, and tissue elasticity observed in field plants. Although certain alterations of leaf structure were expected to coincide with the observed changes and species-specific differences in symplasmic water content and tissue elasticity, these could not be detected by measurements of specific leaf weight or the ratio of dry matter to saturated water content.