Volume contents volume 38 (1985)

Isolated microspores from six cultivars of Brassica napus and one of B. carinata were cultured in modified Nitsch and Nitsch (NN) medium supplemented with 13% (W/V) sucrose, 0.05 mg/l benzyladenine (BA) and 1.00 mg/l nahpthaleneacetic acid (NAA). Embryogenic responses were observed at cultured temperatures ranging from 22 to 32°C. For most genotypes tested, the highest frequency of embryos occurred at 30°C and 7–54 embryos per anther (approx. 17 000 microspores per anther) developed. Although incubation at 30°C produced the highest frequency of embryos, lower culture temperatures induced better quality embryos. A split temperature culture regime of incubation at 32°C for 3 days followed by incubation at 25°C resulted in both high embryo yields and a high percentage of normal embryos. Plantlet development from microspore-derived embryos appeared to be influenced by both genotype and medium.     

Survival,haemolymph osmolality and tissue water of Penaeus chinensis juveniles acclimated to different salinity and temperature levels

Survival, growth, haemolymph osmolality and tissue water of Penaeus chinensis (Osbeck) juveniles (0.11 ± 0.04 g) were investigated, after they were acclimated to 10, 20, 30 and 40 ppt from 33 ppt for 14 days at 24°C, and then acclimated to 12, 18, 24 and 30°C at each salinity for 14 days. The survival of shrimp was the lowest at 10 ppt and 12°C. Growth of shrimp increased with increased temperature in the range 12–24°C, with no significant difference among four salinity levels at 18, 24 and 30°C. Haemolymph osmolality increased with increased salinity, and decreased with increased temperature. The isosmotic point computed from the linear relationship between haemolymph osmolality and medium osmolality was 664, 632, 629 and 602 mOsm/kg which is equivalent to 25.2, 24.1, 24.0 and 23.1 ppt at 12, 18, 24 and 30°C, respectively. Tissue water decreased with increased medium osmolality and haemolymph osmolality. The slope obtained from the relationship between haemolymph osmolality and medium osmolality indicated that there is an impairment of osmoregulatory ability for the P. chinensis juveniles at 12°C.     

Effect of fatty acids on physical properties of microsomes from isolated perfused rat liver

A computer-centered spectrofluorimeter was used to examine the physicochemical properties of hepatic microsomes and microsomal lipids obtained from isolated rat livers perfused with medium containing palmitate or oleate. The fatty acid composition and degree of unsaturation of the liver microsomal lipids reflected that the fatty acid present in the perfusate. The absorption corrected fluorescence, relative fluorescence efficiency, polarization, and fluorescence anisotropy of several fluorescent probe molecules were measured to determine if their different microenvironments may be altered by the type of fatty acid infused. The probe molecules β-parinaric acid and 1.6-diphenyl-1,3,5-hexatriene had higher values for each of these parameters when incorporated into microsomes obtained from livers perfused with a medium containing palmitate than with oleate. The same parameters measured for cholesta-5,7,9(11)-trien-3β-ol and N-phenyl-1-naphthylamine were not altered. These differences appeared to be primarily due to alterations in microviscosity of the probe microenvironments since the rotational correlation time of 1,6-diphenyl-1,3,5-hexatriene was 25% lower in the microsomes from livers perfused with oleate as compared to livers perfused with palmitate. Thermal discontinuities in Arrhenius plots were noted in the intact microsomes but not in the isolated microsomal lipids with the fluorescence probe molecule β-parinaric acid. Break points occurred at 10°C and 26°C for microsomes from livers perfused with palmitate and at 12°C and 17°C for microsomes from livers perfused with oleate containing medium. These results suggest that the physicochemical properties of liver microsomes were determined in part by the fatty acid in the perfusate.     

The in vitro biosynthesis and secretion of glycerol by larval fat bodies of chilled Ostrinia nubilalis

Fat bodies from diapausing fifth-instar larvae of Ostrinia nubilalis were incubated in vitro at 5 or 23°C in Grace's medium and the glycerol contents of the organ and incubation medium determined. Fat bodies from diapausing larvae chilled 3 weeks at 5°C secreted glycerol into the medium at 5°C at a net rate of approx. 0.75 nmol/mg fat body dry wt/h for at least 96 h while the tissue levels remained essentially constant. Depending upon the experiment, from 6 to 15 times more glycerol was produced in 24 h at 5°C by these fat bodies than by those taken from diapausing unchilled larvae and incubated at either 5 or 23°C. A minimal chilling period of 10–12 days was recognized as necessary for chilled larval fat bodies to demonstrate rates of glycerol synthesis greater than those of unchilled larvae and the lag showed a temporal correlation with changes in haemolymph glycerol concentrations. These results suggest that this response to chilling by O. nubilalis is relatively slow. While incubation, at 23°C, of fat bodies from previously chilled larvae did not result in cessation of glycerol secretion, the rate of its appearance in the culture medium decreased during the 24-h incubation period. Although the ability of chilled fifth-instar larvae to accumulate glycerol is not dependent upon the diapause state results show that clearance of glycerol from the haemolymph by rewarmed O. nubilalis is related to diapause intensity.     

In vitro culture of Plasmodium yoelii blood stages

Lewis-Hughes P. H. and Howell M. J. 1984. In vitro culture of Plasmodium yoelii blood stages. International Journal for Parasitology14: 447–451. Plasmodium yoelii infected reticulocytes were cultured for 72 h at either 37 or 20°C in MEM (Eagle's modification) medium containing, in addition, glucose, para-aminobenzoic acid and 5% foetal calf serum, buffered at pH 7.3 with sodium bicarbonate/ HEPES and maintained under 10% CO₂ in air. Red blood cell numbers were more stable at 20°C than at 37°C. Culture at both temperatures resulted in an increase in parasitaemia of the reticulocyte population over the initial 36 h at 37°C and for at least 72 h at 20°C. The effects of different temperatures appeared to be related to the continued presence of target cells. Parasites were not detected after 72 h culture at 37°C, but persisted for up to 120 h at 20°C. Increasing parasitaemia at both temperatures was associated with changes in the numbers of some parasite development types. Early falls in schizont numbers were associated with an increase in the numbers of ring forms. Trophozoite numbers tended to remain constant throughout the culture period. Viability of parasites cultured for 36 h was confirmed by their infectivity to CBA mice. In addition, parasites progressively incorporated H³-leucine into TCA-precipitable material over the initial 36 h of culture.     

Development rate,number of mature oocytes at emergence and adult size ofEncarsia tricolor at constant and variable temperatures

The duration of the development of the aphelinidEncarsia tricolor Föerster (a parasitoid of the aleyrodidTrialeurodes vaporariorum (Westwood), adult size and number of mature oocytes at emergence were determined under constant and variable temperature regimes. Females developed successfully from 14 to 32°C, but a 100% of pupal mortality was observed at 34°C. Males developed successfully from 16 to 28°C and they developed faster than females. Female and male, egg to adult development at constant temperatures ranged from 51.1 (14°C) to 14.3 (28°C) days and from 32.6 (16°C) to 11.8 (28°C) days, respectively. Predictions of the rate of development at variable temperatures were more accurate when made from 2nd and 3rd degree polynomials than from linear regressions. The number of mature oocytes at emergence ranged from 0.1 (30°C) to 2.2 (20°C). FemaleE. tricolor attained the greatest size at 20–24°C. The comparison with literature data shows thatE. tricolor develops faster thanT. vaporariorum at temperatures above 15°C.     

The induction of collagenase by thyroxine in resorbing tadpole tailfin in vitro.

Explants of tailfin tissue from Rana pipiens or R. catesbiana undergo complete reepithelialization of the cut surfaces (healing) in culture at 22°C. After reepithelialization, these healed explants maintain normal tissue organization in subsequent culture at either 22 or 37°C. When thyroxine is added to the medium of these healed explants, an organized resorption of the tissue occurs, characterized by a gradual loss of explant size and the loss of tissue collagen which is concomitant with the appearance of collagenase in the medium. These throxine-dependent changes occur at 22°C and, more rapidly, at 37°C. Control reepithelialized explants do not resorb, lose collagen or produce collagenase. In contrast, tailfin tissue from both species, when placed in culture at 37°C directly, fails to reepithelialize and undergoes massive resorption, independent of hormonal conditions. These findings indicate that collagenase is involved in the physiological removal of collagen from the resorbing tadpole tailfin and that the expression of collagenase activity is regulated by thyroxine.     

Expression and characterization of a thermostable penicillin G acylase from an environmental metagenomic library

One clone (ACPGA001) exhibiting penicillin G acylase (PGA) activity was screened from a metagenomic library by using a medium containing penicillin G. A novel PGA gene from the inserted fragment of ACPGA001 was obtained by sequencing. The amino acid sequence of ACPGA001 PGA exhibited <33 % similarity to PGAs retrieved from GenBank. This gene was expressed in Escherichia coli M15 and the recombinant protein was purified and characterized. The ACPGA001 PGA exhibited a maximum activity at 60 °C and showed high activity at pH 4–10 with an optimum pH of 8.0. This enzyme was stable at 40 °C for 70 min with a half-life of 60 min at 55 °C. These beneficial characteristics of ACPGA001 PGA provide some advantages for the potential application of ACPGA001 PGA in industry.     

Incubation temperature critical to successful stimulation of in vitro zygotic embryo growth in four Australian native Cyperaceae species

Many species of Western Australian Cyperaceae (sedges) are vital components of the indigenous flora but commonly display low seed set, poor seed quality and intractable seed dormancy. We report the effects of incubation temperature and in vitro growth media on whole seed germination compared with extracted zygotic embryo growth in Tetraria capillaris, T. octandra, Lepidosperma drummondii and L. tenue. No germination was observed from intact whole seeds of all test species regardless of the treatment evaluated. In contrast, excised zygotic embryos of all study species exhibited significant increases in growth when cultured at 15°C compared to embryos incubated at 25°C; however, optimal media for embryo growth were genera specific. Extracted embryos of T. capillaris and T. octandra exhibited maximum percentage growth (30 and 40%, respectively) at 15°C on ½ MS medium with no plant growth regulators required. In the case of L. drummondii and L. tenue 1 μM thidiazuron was a necessary addition to the ½ MS medium resulting in 40 and 77% growth of embryos (at 15°C), respectively. Incubation of extracted embryos at 25°C (regardless of medium treatment) resulted in <10% embryo growth for T. octandra and L. tenue, while the remaining two species (L. drummondii, T. capillaris) showed no embryo growth at 25°C on any medium treatment.     

Germination responses of four native terrestrial orchids from south-west Western Australia to temperature and light treatments

We report an investigation into the impact of temperature and illumination on in vitro symbiotic and asymbiotic germination of the threatened taxon Caladenia huegelii, and three other orchid spp. namely—Caladenia latifolia, Microtis media and Pterostylis sanguinea, all species from south-west Western Australia, a recognized biodiversity hotspot. High symbiotic germination on oatmeal agar (OMA + fungal symbionts specific to each species) was recorded in three species in continuous dark incubation i.e. C. huegelii seeds (98 % germination at 25 °C), and M. media and P. sanguinea (93 and 98 % respectively at 20 °C). Highest symbiotic germination for C. latifolia (100 %) was observed at 15 and 20 °C under light treatment (12/12 h light/dark). Low temperature incubation (10 °C) significantly suppressed symbiotic germination/development of seedlings across all species. Asymbiotic media treatments assessed (OMA minus fungal symbionts, Pa₅ and ½ MS), failed to stimulate any germination with C. latifolia seeds at 20 °C in either light or dark treatments after an 8 week incubation period. Seeds of M. media sown onto ½ MS medium resulted in higher germination in all developmental stages (3–5) in dark treatment than OMA and Pa₅. Seeds of P. sanguinea sown onto ½ MS medium resulted in higher overall germination in all developmental stages (3–5) in light and dark incubation compared to OMA and Pa₅. OMA supported the highest asymbiotic germination (100 %) in both light and dark incubation with M. media (only to stage 3) but did not support germination and development with other spp. tested. Caladenia huegelii seeds reached developmental Stage 3 (i.e. germinated), but only on Pa₅ medium and only at a relatively low rate in either light (2.6 %) or dark (2.1 %). Germination was higher and development of seedlings faster overall in all test species in symbiotic compared with asymbiotic media treatments. P. sanguinea seeds demonstrated the best response (among species tested) to asymbiotic germination on ½ MS with 40–53 % of germinated seeds spread over developmental stages 3–5 in light or dark incubation (at 20 °C) respectively. Illumination had no effect on fungal symbiont growth across all species, however incubation temperature treatments (10, 15, 20 and 25 °C) affected fungal growth rate. Growth of the fungal symbionts of C. huegelii, M. media and C. latifolia demonstrated significantly lower activity at 10 °C, but the cumulative radial growth rate of the P. sanguinea fungal symbiont reached 64 cm² after only 2 weeks at all temperatures tested, including 10 °C. The study highlights differences in symbiotic and aysmbiotic germination and early protocorm development in vitro between co-occurring herbaceous terrestrial Australian orchid taxa in response to variations in basal media, temperature and light.     

Amino acid accumulation in frog muscle: I. Steady-state glycine accumulation at o °C

Glycine is not significantly metabolized by frog muscle maintained at o °C in vitro. Nevertheless, in this preparation steady-state levels of [¹⁴C]glycine as high as 20 times the external concentration are attained after 3–6 days at o °C. The concentration gradient at the steady state depends on the external concentration, being highest at low external concentrations (approx. 0.1 mM) and reversed at external concentrations above 10 mM.A plot of the steady-state cellular levels of glycine vs the external concentration reveal linear and saturable components. The linear fraction has an average distribution ratio of 0.54 indicating that glycine is partially excluded from the muscle water at this temperature.Efflux of labeled glycine at o °C from previously loaded frog muscle follows first-order kinetics. The rate constant increases with increasing concentrations of glycine in the external medium (efflux facilitation).The steady-state results are shown to be consistent with an adsorption model for amino acid accumulation as well as a model in which amino acid enters the cell via a carrier and exits via a bidirectional leakage pathway. A model in which efflux proceeds through the carrier does not fit the data. This indicates that an alternative to exchange diffusion is needed to explain the observed efflux facilitation.     

Preservation of energy-linked functions of insect mitochondria by simple freezing methods.

Attempts are made to preserve some energy-linked functions of mitochondria isolated from the fat body of the cockroach Nauphoeta cinerea. The ability of mitochondria to sustain appreciable levels of oxidation, phosphorylation, respiratory control and 2, 4-DNP-stimulation of respiration which are normally lost during incubation at 30°C for 2 hrs or during aging at 0–2°C for 48 hrs can be preserved effectively by storing mitochondrial suspensions in sucrose - EDTA medium for 1 week at - 20°C and at least 3 weeks at -196°C (liquid nitrogen temperature). DMSO (dimethyl sulfoxide; 10% v/v) was found to be ineffective as an aid to preservation at 0–2°C, a significant aid at -20°C and unnecessary at -196°C. With slight variations (for flight muscle mitochondria which requires DMSO at -196°C), this procedure is also effective for mitochondria isolated from other tissues of various insect species. EDTA was found to be an essential ingredient of the isolation medium and therefore for all storage procedures. Both lyophilization and the preincubation of mitochondria with nupercaine (400 μM) have deleterious effects.     

Conditional rifampicin sensitivity of arif mutant ofEscherichia coli: rifampicin induced changes in transcription specificity

Arif mutantof Escherichia coli that exhibits medium and temperature-dependent sensitivity to rifampicin is described. In the absence of rifampicin, this strain grows in minimal and rich media at 30°C and 42°C. In its presence it is viable in rich medium at both temperatures, but in minimal medium only at 30°C. In minimal-rifampicin medium at the higher temperature, RNA synthesis is decreased. The addition of certain divalent salts (MgSO₄, CaCl₂, BaCl₂) in excess, or chelators (EDTA, EGTA, o-phenanthrolein) greatly increase viability in minimal-rifampicin medium at 42°C. Excess MgSO₄ (10 mM) also increases the rate of RNA synthesis in the same medium. A model is proposed wherein therif mutation is suggested to cause a structural change in RNA polymerase that allows the binding of rifampicin and other ligands at 42°C. Rifampicin-binding is suggested to alter the conformation of RNA polymerase, impairing its ability to express genes required for growth in minimal medium. Implicit in this view is the assumption that these genes are structurally different from those expressed in rich medium in respect of certain template features recognized by RNA polymerase.     

Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda)

Fried B. and Huffman J. E. 1982. Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda). International Journal for Parasitology12: 427–431. Encysted metacercariae of Sphaeridiotrema globulus were obtained from naturally infected Goniobasis virginica snails in Morris County, New Jersey, U.S.A. These metacercariae were successfully excysted within l h in an alkaline bile salts trypsin medium maintained at 41°C in the absence of acid pepsin pretreatment. Encysted metacercariae treated in 3% sodium bicarbonate produced successful infections in day-old domestic chicks and worms became ovigerous in the lower ileum by day 4. Excysted metacercariae transferred to the chorioallantois of 6–10-day-old chick embryos maintained at 41°C developed into ovigerous adults by day 4.     

Temperature-dependent development, diapause and cold tolerance of Gratiana graminea, a potential biological control agent of Solanum viarum in Florida, USA

The objectives of this study were to examine temperature-dependent development, diapause and cold tolerance of Gratiana graminea Klug (Chrysomelidae), a candidate biological control agent of tropical soda apple, Solanum viarum Dunal (Solanaceae). Immature development was examined at six constant temperatures ranging from 15°C to 30°C. Diapause induction was determined by exposing adults to either long or short photoperiods at 20°C and cold tolerance was assessed by exposing adults to 0°C. G. graminea completed development at temperatures ranging from 20°C to 30°C. Linear regression estimated a lower temperature threshold of 11.7°C and 312 degree-days were required to complete development. Diapause was induced when adults were exposed to short photoperiods (10:14 L:D h) at 20°C. The lethal times for diapausing adults of G. graminea at 0°C (LT₅₀?=?19?days, LT₉₀?=?41?days) were two times higher compared to Gratiana boliviana Spaeth, a biological control agent already established in south and central Florida, USA. The presence of diapause and the greater cold tolerance suggest that G. graminea may establish and perform better than G. boliviana in northern Florida.     

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.     

A new pilot plant scale acetifier designed for vinegar production in Sub-Saharan Africa

A novel thermotolerant strain Acetobacter senegalensis sp. nov. (CWBI-B418^T) isolated in Senegal from mango fruit, previously freeze-dried and conserved at 4 °C under vacuum packaging was successfully rehydrated into an acetifying medium. It was used as an inoculum culture and then applied into a new pilot plant scale acetifier (300 L) for vinegar production. This latter was specifically designed to produce a high volume and quality of vinegar in Sub-Saharan Africa at fermentation temperature of 35 °C. Several semi-continuous cycles of acetic acid fermentations were carried out. The behaviour of substrate and product concentrations, population of bacteria into the reactor was analysed as well as the evolution of acidity, acetification rates and stoichiometric yields. Operation with this novel bioreactor allowed achieving 8% (v/v) of acetic acid concentration at 35 °C.     

Conducting High acetic acid and temperature acetification processes by Acetobacter pasteurianus UMCC 2951

In this study Acetobacter pasteurianus strain UMCC 2951 was tested as a microbial starter to conduct acetification processes by repeatedly cultivation cycles under high temperature acetification at 40 ± 1 °C. Acid production and acetification rate increased with repeated cultures under high temperature acetification as adaptation period increased, but were still lower than acetification at 30 ± 1 °C. However, the addition of 0.15 % calcium chloride reduced the negative effects of 40 ± 1 °C on both acid production and acetification rate compared to 30 ± 1 °C. A strong decrease in fatty acids and phosphatidylethanolamine and increases in phosphatidylcholine and phosphatidylglycerol in cell membranes were found under high acid and high temperature acetification. In addition, transmission electron microscope images reveal a more compact cell wall when calcium chloride was added to the cultivation medium. The strategy used in this study confirmed that the use of acetic acid bacteria as microbial starters could be effective also at temperature above the optimal values, when acetification processes are managed through repeated semi-continuous cycles.     

Production of Extracellular Acid Proteases by Aspergillus Clavatus

The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 °C. The highest acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1%(w/v) at 30 °C at the third day of incubation. Cultures developed in Vogel medium with glucose at 2%(w/v) showed at about 45% of proteolytic activity when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 30, 10 and 5 min, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35?°C for a few days and by then incubating them in the dark at 25?°C. Pre-culturing anthers at 35?°C for 4?days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15?°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.