Spontaneous chlorophyll mutants of Pennisetum americanum: Genetics and chlorophyll quantities

Summary Thirteen spontaneously occurring chlorophyll deficient phenotypes have been described and their genetic basis was established. Ten of these — white, white tipped green, patchy white, white virescent, white striping 1, white striping 2, white striping 4, fine striping, chlorina and yellow virescent showed monogenic recessive inheritance and the remaining three — yellow striping, yellow green and light green seedling phenotypes showed digenic recessive inheritance. The genes for (i) white tipped green (wr) and yellow virescent (yv) and (ii) patchy white (pw) and white striping 1 (wst 1) showed independent assortment. Further, the genes for white (w), white tipped green (wr) and yellow virescent (yv) were inherited independently of the gene for hairy leaf margin (Hm).In the mutants — white tipped green, patchy white, white striping 1, white striping 2, fine striping, chlorina, yellow virescent, yellow striping, yellow green and light green phenotypes total quantity of chlorophyll was significantly less than that in the corresponding controls, while in white virescent there was no reduction in the mature stage. For nine of the mutants the quantity of chlorophyll was also estimated in F₁'s (mutant x control green). In F₁'s of six of the mutants — white tip, patchy white, chlorina, yellow virescent, fine striping and yellow striping the quantity of chlorophyll was almost equal to the wild type. In the F₁'s of three of the mutants — white striping 1, white striping 2 and light green an intermediate value between the mutant and wild types was observed. In yellow virescent retarded synthesis of chlorophyll, particularly chlorophyll a was observed in the juvenile stage. Reduced quantity of chlorophyll was associated with defective chloroplasts. In the mutants — white tipped green, white virescent, fine striping, chlorina, yellow striping, yellow green and light green defective plastids were also observed. In patchy white secondary destruction of chlorophylls and the presence of defective plastids were found to be associated with reduced chlorophyll quantity at maturity.Paper chromatographic studies of leaf flavonoids revealed some variation between the inbreds, but there were three common spots, 7, 8 and 9, except for PDP in which the spot 8 was absent. Chlorophyll deficient mutants differed from their respective controls in the absence of one or more of the spots present in the controls and in the presence of new spots in some of the mutants.Most of the chlorophyll mutants showed higher survival rate in the Kharif season than in Rabi season which was attributed to the higher mean day temperature and longer day light period in the Kharif season than in Rabi season.     

Differential Expression and Association of Calcium Channel Subunits in Development and Disease

Voltage-gated calcium channels (VDCC) are essential to neuronal maturation and differentiation. It is believed that important signaling information is encoded by VDCC-mediated calcium influx that has both spatial and temporal components. VDCC are multimeric complexes comprised of a pore-forming ₁ subunit and auxiliary and ₂/ subunits. Changes in the fractional contribution of distinct calcium conductances to the total calcium current have been noted in developing and differentiating neurons. These changes are anticipated to reflect the differential expression and localization of the pore-forming ₁ subunits. However, as in vitro studies have established that regulates the channel properties and targeting of ₁, attention has been directed toward the developmental expression and assembly of isoforms. Recently, changes in the component of the omega-conotoxin GVIA (CTX)-sensitive N-type VDCC have indicated differential assembly of _1B with in postnatal rat brain. In addition, unique properties of 4 have been noted with respect to its temporal pattern of expression and incorporation into N-type VDCC complexes. Therefore, the expression and assembly of specific ₁/ complexes may reflect an elaborate cellular strategy for regulating VDCC diversity. The importance of these developmental findings is bolstered by a recent study which identified mutations in the 4 as the molecular defect in the mutant epileptic mouse (lethargic; lh/lh). As 4 is normally expressed in both forebrain and cerebellum, one may consider the impact of the loss of 4 upon VDCC assembly and activity. The importance of the lb and 4 isoforms to calcium channel maturation and assembly is discussed.     

Organic solvents for bioorganic synthesis

Summary The influence of solvents on enzymatic activity and stability was investigated. As a model reaction the -chymotrypsin-catalyzed esterification of N-acetyl-l-phenylalanine with ethanol was used. The enzyme was adsorbed on porous glass beads and used in various solvents. Small amounts of water were added to increase the enzymatic activity. These enzyme preparations obeyed. Michaelis-Menten kinetics. K _m,app decreased slightly with the log P value of the solvent while V _app increased markedly with the log P value. Log P values were also useful for generalizing the influence of solvents on enzyme stability. The enzyme preparations showed a markedly higher thermostability in dry solvents having log P values >0.7 than in less hydrophobic solvents.Also the operational stability was better in the more hydrophobic solvents. The amount of water added to the enzyme preparations greatly influenced the initial reaction rates. For some solvents optimal water contents were determined. The thermostability decreased with increasing water content.The observations are summarized in the conclusion that more hydrophobic solvents are preferable to less hydrophobic ones. The log P value gives a good guidance when selecting an organic solvent for enzymatic conversions.     

Quantitative comparisons between the karyotypes of Australian marsupials from three different superfamilies

Relative DNA values and percent lengths of chromosome arms have been studied for six species of the super-family Dasyuroidea, five species of Phalangeroidea and four species of Perameloidea. By multiplying by relative DNA values all percent lengths have been expressed in the same units. Two species are said to share a chromosome if neither of the arms differs at the 5% level of probability. It is argued that if species share at least two two-armed chromosomes, this can be taken as evidence of relationship. A standard dasyurid karyotype has been defined and individual species of Dasyuridae show only small deviations from it. The nature and significance of the bi-modal distribution of chromosome numbers in marsupials is discussed.     

Production of multiple wheat-rye 1RS translocation stocks and genetic analysis of LMW subunits of glutenin and gliadins in wheats using these stocks

Summary A triple (1AL.1RS/1BL.1RS/1DL.1RS) and three double (1AL.1RS/1BL.1RS, 1AL.1RS/1DL.1RS, 1BL.1RS/1DL.1RS) wheat-rye 1RS translocation stocks were isolated from a segregating population using the Gli-1, Tri-1 and Sec-1 seed proteins as genetic markers. These stocks carried 42 chromosomes and formed the expected multivalents (frequency of 14–25%) at metaphase 1. They gave floret fertility ranging from 40–60%. These stocks were subsequently used to determine the genetic control of low-molecular-weight (LMW) glutenin subunits in Chinese Spring and Gabo by means of two-step one-dimensional SDS-PAGE. All of the B subunits and most of the C subunits of glutenin were shown to be controlled by genes on the short arms of group-1 chromosomes in these wheats. The other C subunits were not controlled by group-1 chromosomes. The triple translocation line served as a suitable third parent in producing test-cross seeds for studying the inheritance of the LMW glutenin subunits and gliadins in wheat cultivars, e.g. Chinese Spring and Orca. The segregation patterns of the LMW glutenin subunits in these cultivars revealed that the subunits were inherited in clusters and that their controlling genes (Glu-3) were tightly linked with those controlling gliadins (Gli-1). The LMW glutenin patterns d, d and e in Orca segregated as alternatives to the patterns a, a and a in Chinese Spring controlled by Glu-A3, Glu-B3 and Glu-D3 loci on chromosome arms 1AS, 1BS and 1DS, respectively, thus indicating that these patterns were controlled by allelic genes at these loci.     

Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

Enhanced production of scopolin bySolanum aviculare cells immobilised within Ca-alginate gel beads

Summary Different matrices, obtained by varying calcium (0.1 to 1.5M) and alginate (1 to 1.5%) concentrations, were used to study the influence of immobilisation parameters on the behaviour ofS. aviculare. A significant modulation of cell growth, cell release, and scopolin production and excretion has been observed. Physiological and morphological characteristics ofSolanum aviculare cells immobilised within Ca-alginate beads were notably different from those of suspended cells. ImmobilisedS. aviculare have accumulated scopolin (up to 120 g·g^–1 FWB) within beads and excreted it into the culture medium (up to 8 g·g^–1 FWB). Contrary to suspended cells which have accumulated only traces of this metabolite within intracellular compartments (1 g·g^–1 FWB), no scopolin has been found into the culture medium.Abbreviations ANA -Naphthaleneacetic acid - HPLC high performance liquid chromatography - FWB fresh weight biomass - LS medium Linsmaier and Skoog medium - MS mass spectroscopy - NMR nuclear magnetic resonance - r² coefficient of determination - s standard deviation     

Filamentous Marine Fungi as Producers of <Emphasis Type="Italic">O</Emphasis>-Glycosylhydrolases: β-1,3-Glucanase from <Emphasis Type="Italic">Chaetomium indicum</Emphasis>

Ninety fungal strains (42 species) isolated from marine habitats were studied for their ability to produce extracellular enzymes. Cultural filtrates of these strains were shown to contain a series of glycosidases (-glucosidases, N-acetyl--glucosaminidases, -galactosidases -mannosidases) and glucanases (1,3--glucanases, amylases) which varied with habitat. The level of activity depended on the species of fungi. Several promising strains capable of producing both individual enzymes and a set of enzymes for splitting carbohydrate-containing compound have been isolated. Optimal conditions for growth of Chaetomium indicum and for biosynthesis of -1,3-glucanase were determined. -1,3-Glucanase was isolated using ion-exchange chromatography, ultrafiltration, and gel filtration. The presence of 2 enzyme forms was shown; both forms were exo--1,3-glucanases.     

Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion

The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca²⁺ (EC₅₀=2.4 M). The stable GTP analogue GTPS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca²⁺ concentrations. At optimal Ca²⁺ (10 M) the effect of GTPS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A₂. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca²⁺ dependent effects, (ii) phospholipase C was not activated in the absence of Ca²⁺ and insulin secretion due to the phorbol ester TPA displayed a different Ca²⁺ dependency, (iii) arachidonic acid did not elicit Ca²⁺ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca²⁺ or TPA is attenuated by the inhibitory guanine nucleotide GDPS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.Abbreviations InsP₃ inositol trisphosphate - Ptd-InsP₂ phosphatidylinositol 4,5-bisphosphate - GTPS guanosine 5-(3-O-thio)triphosphate - GDPS guanosine 5-(2-O-thio)diphosphate - Gpp(NH)p guanyl-5-yl imidodiphosphate - TPA 12-O-tetradecanoylphorbol-13-acetate - OAG 1-oleoyl-2-acetylglycerol - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA (ethylenebis(oxyethylenenitrilo)tetraacetic acid - DAG diacylglycerol - [Ca²⁺]_i cytosolic free Ca²⁺ concentration     

Triggering and predisposing factors in the “Red” decline syndrome of Norway spruce (Picea abies)

Summary Analysis of 62 mature Norway spruce (Picea abies provenance Viborg) trees growing in a Danish plantation was undertaken along with analysis of their nutrient contents (N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B and Na), in each of the three youngest needle age classes, from branches of four exposure directions near the tree top. The aim was to investigate if one among the studied possible predisposing factors was also a triggering factor in the 1989 outbreak of the Red Norway spruce decline in Denmark. Neither nutrient imbalance or deficiency, nor excessive N-deposition or salt-stress were indicated as triggering factors in 1989. The Red syndrome, noticeable for the bright red colour of the current-year needles, was found to be an extension of the European type Novel Decline. Red syndrome is similar to previously reported phenomena of top-dying and sub top-dying, in that it had fewer needle age classes and significantly higher contents of mobile cations (and Ca) in the younger needle classes. Tree ring analysis suggested that the Red syndrome was initiated in the early 1980s, when the trees experienced adverse climatic conditions. Because of this long-term development of the Red Norway spruce decline syndrome, it is concluded that a triggering factor is of minor importance relative to the multitude of predisposing factors.     

Beobachtungen zu Biologie und Verhalten des Gro?en Kragenlaubenvogels (Chlamydera nuchalis)

Zusammenfassung Von Mai bis Juli 1969 wurde im Gebiet von Darwin und Arnhemland (Northern Territory, Australien) Biologie und Verhalten vonChlamydera nuchalis studiert.28 Lauben wurden gefunden. Einzelheiten des Laubenbaues und der hierzu verwendeten Materialien werden beschrieben. Das muß etwa 4–5000 Zweige zum Laubenplatz schaffen; die Wände der Laube bestehen aus etwa 1400 bis 1840 Zweigen.Die zur Ausschmückung der Laube verwendeten Gegenstände umfaßten Molluskenschalen, Knochen, Glasstückchen, Steine und kleine glänzende Metallobjekte. Das Gesamtgewicht des Laubenschmuckes betrug 6,6 bis 12,1 kg. Größe und Gewicht der Einzelobjekte schwankten zwischen 4–10 mm und 0,2–1,2 g bzw. 73 × 36 mm und 40 g. Ihre Gesamtzahl liegt zwischen 5000 und 12 000. Bevorzugt werden weiße und graue Objekte; auch grüne Glasstückchen wurden gefunden.Die Technik des Laubenbaus, die ausschließlich das ausführt, wird beschrieben. Der Rohbau nimmt etwa 3 Wochen in Anspruch, doch werden im Anschluß daran dauernd neue Objekte eingetragen. Anzeichen für das Ausmalen der Laube wurden nicht gefunden. Während der Balz singt das intensiv auf Bäumen oder am Boden. Die einzelnen Balzphasen werden beschrieben. Während des Höhepunktes versucht das durch Sprünge im Kreis das an der Laube zu halten. Begattung wurde außerhalb der Laube nahe dem Ausgang beobachtet. Offensichtlich hat ein mehrere während einer Balzperiode. Während der Balz wird die Laube dauernd ausgebessert und ihr Schmuck neu arrangiert.

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On the biology and behaviour of the Great Grey Bower Bird(Chlamydera nuchalis)

Summary From May to July, 1969 the author observed in the region of Darwin and Arnhem Land (Northern Territory, Australia) the biology and behaviour of the Great Grey Bower Bird.28 bowers were found, in three cases the building process was observed. Bower-building starts with cleaning up an area of 210 to 95 cm and with forming a 7.5 cm thick mat of small twigs intertwined in all directions. The walls are made of uniform twigs the measurements of which are on an average 29.2 mm in length and 2.4 mm in thickness at the lower end. A wall consists of 700 to 920 twigs placed parallel to each other (1400 to 1840 twigs on the two walls). The carries 4000 to 5000 twigs to the site of the bower. The paper gives the measurements of the bower and its orientation towards cardinal points.The bower is completed by a collection of ornaments which consists of land and sea shells of the generaXanthomelon, Venus andTelescopium, bone and glass fragments, stones (almost exclusively quartzite and limestone), and small pieces of glossy metal objects, tin seals etc. The lowest total weight of all objects collected was 6.2 kg, the highest was 12.1 kg. The size and weight of the objects varies between 4 to 10 mm and 0.2 to 1.2 g and 73 by 36 mm and 40 g respectively.Their total number was established at 5000 to 12000.Chlamydera nuchalis prefers white and grey objects with the exception of glass of which also green fragments were found. Some objects were experimentally painted blue, red, black, yellow and green. The discarded them successively and threw them out of the bower.The paper describes the technique of bower-building which is carried out solely by the . It takes the bird three weeks to finish the construction in the rough, but even afterwards the systematically continues to complete the bower, adding a growing number of ornamental objects. During the display period, however, a rebuilt a bower, which had been destroyed, within 2.5 days. The activities observed are shown in the table. Bower Birds may carry, on an average, 90 twigs and 28 glass fragments per hour to the bower. The bower wall painting behaviour ofChlamydera maculata andPtilonorhynchus violacaeus was not observed inChlamydera nuchalis. No traces of painting were found in any of the bowers observed. The author found an interesting behaviour pattern in two who stuck pieces of food into the cracks in the walls. This behaviour strikingly resembles that of passerines feeding their young.During the display period the sings ardently either on trees or on the ground. Display consists of alluring manifestations of the , including nape-crest presentation and bounds. At the bower, the shows the some objects on the opposite side of the passage, and after bowing and raising his head, he turns it so that the nape is directed toward the , and presents to her its lilac crest. During the culminating phase of display the attempts, by circling in leaps, to keep the at the bower. Mating was observed outside the bower, next to its exit. A apparently has several females during the display period. Also during display the rearranges and completes the bower.

     

Diagnosing lactic acid fermentation based on specific rates of growth,substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols 1/h specific rate of cell growth - 1/h specific rate of substrate consumption - 1/h specific rate of product formation - ^* dimensionless specific rate of cell growth - ^* dimensionless specific rate of substrate consumption - ^* dimensionless specific rate of product formation - _max 1/h maximum specific rate of cell growth - _max 1/h maximum specific rate of substrate consumption - _max 1/h maximum specific rate of product formation - X g/l cell mass concentration - S g/l substrate concentration - S ^* dimensionless substrate concentration - S ₀ g/l initial substrate concentration - P g/l product concentration     

Amino acid and protein oxidation in cardiovascular disease

Summary. Substantial evidence suggests that oxidative events contribute to the pathogenesis of atherosclerotic heart disease. For example, animal model data and numerous in vitro studies point to specific pathways as participants in disease initiation and progression. Moreover, recent clinical studies demonstrate clinical utility in monitoring systemic levels of protein-bound nitrotyrosine as a predictor of risk for coronary artery disease, atherosclerotic burden, and response to statin therapy. However, a definitive cause-and-effect relationship between oxidation and atherosclerosis has yet to be established, and multiple recent large prospective antioxidant intervention trials have failed to significantly impact upon disease risk and progression. In this review we highlight why such failures should not be taken as an indictment of the Oxidation Hypothesis. Emphasis will be placed on discussion of molecular markers whose structures convey information about oxidation pathways leading to their formation, and which appear to be mechanistically linked to the disease process. Only through rational design of targeted interventions aimed at suppressing distinct oxidation pathways, with concomitant monitoring of antioxidant efficacy in human clinical studies, will answers to the role of oxidation in the pathogenesis of human atherosclerosis be defined.     

Comparison of the activity of immobilised and freely suspended Streptomyces coelicolor A3(2)

Streptomyces coelicolor was immobilised naturally in porous support materials and its growth, glucose uptake and actinorhodin production were compared with freely suspended culture using defined and complex media. When the defined medium was used, the most pronounced difference between the two cultures was the accumulation of actinorhodin extracellulary in freely suspended and intracellularly in immobilised cultures. In the complex medium, however, actinorhodin was excreted by both cultures. In addition, the complex medium yielded 50 times as much actinorhodin compared to the defined medium. Further increases in product concentration were obtained by repeated batches of immobilised culture, which showed stability for at least 3 months.     

Effect of biocatalyst activity changes in continuous reactions in the gas phase

Summary In the gas phase bioreactions, continuous production rate depends on the biocatalyst activity and complete dehydration causes the biocatalyst to lose most of its activity. To overcome these difficulties, a theoretical method is suggested along with the new design of biocatalyst. This will be applicable and helpful for the optimization of the gas phase continuous bioreaction.Nomenclature C_A ethanol concentration [mol/mL] - C_P acetaldehyde concentration [mol/mL] - X_P acetaldehyde composition     

Observation of the chemical structure of fructooligosaccharide produced by an enzyme fromAureobasidium sp. ATCC 20524

The chemical structure of the oligosaccharide produced from sucrose by an enzyme extracted fromAureobasidium sp. ATCC 20524 was observed. The GC-MS analysis by methylation indicated that this oligosaccharide is composed of 2-linked, 1,2-linked fructose and 1-linked glucose. The [¹³C]-NMR spectrum indicated that the 1,2-linked glycosidic linkages of fructose of this oligosaccharide are, and the 1-linked glycosidic linkages of fructose are . This investigation suggested that this oligosaccharide is isokestose.     

Reduced expression of a distal gene of the prn gene cluster in deletion mutants of Aspergillus nidulans: genetic evidence for a dicistronic messenger in an eukaryote.

Summary Temperature sensitive dnaAts46 mutants, in which initiation of chromosome replication is blocked at 42° C, are unable to maintain a dv plasmid at the permissive temperature unless the plasmid carries a mutation in gene P of the type permitting phage to grow in groP (dnaB) bacteria. The growth rate of dnaAts46 mutants seems to be impaired by the presence of the dvP mutant plasmid.Cold sensitive dnaAcos mutants which overinitiate replication at low temperature and grow normally only at 40° and above, can maintain efficiently dvP ⁺ plasmids as well as dvP mutants. Cold sensitivity of dnaAcos mutants is suppressed by the presence of the plasmid dvP ⁺ and by certain dvP mutants, but not by others.The gene P product seems to act by reducing the initiation potential of both types of dnaA mutants, aggravating the initiation defect in dnaAts46 and correcting the overinitiation of dnaAcos.     

Telomeres and P-element of Drosophila melanogaster contain sequences that replicate autonomously in Saccharomyces cerevisiae

Summary We have isolated restriction fragments from a shotgun collection of Drosophila DNA which function as autonomously replicating sequences (ARS) in Saccharomyces cerevisiae and hybridize with telomeric regions of the 2L, 2R, 4, and X chromosomes. In an independently obtained set of D. melanogaster clones five fragments hybridize in situ with telomeres and a number of internal sites. Two of them also contain ARSs. A Drosophila mobile P-element also possesses ARS activity in yeast.     

3D QSAR studies on peroxisome proliferator-activated receptor γ agonists using CoMFA and CoMSIA

The peroxisome proliferator-activated receptors (PPARs) have increasingly become attractive targets for developing novel anti-type 2 diabetic drugs. We employed comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) to study three-dimensional quantitative structure–activity relationship (3D QSAR) based on existing agonists of PPAR (including five thiazolidinediones and 74 tyrosine-based compounds). Predictive 3D QSAR models with conventional r² and cross-validated coefficient (q²) values up to 0.974 and 0.642 for CoMFA and 0.979 and 0.686 for COMSIA were established using the SYBYL package. These models were validated by a test set containing 18 compounds. The CoMFA and CoMSIA field distributions are in general agreement with the structural characteristics of the binding pockets of PPAR, which demonstrates that the 3D QSAR models built here are very useful in predicting activities of novel compounds for activating PPAR.      

Cold tolerance of Littorinidae from southern Africa: intertidal snails are not constrained to freeze tolerance

All intertidal gastropods for which cold tolerance strategies have been assessed have been shown to be freeze tolerant. Thus, freeze tolerance is considered an adaptation to the intertidal environment. We investigated the cold tolerance strategies of three species of subtropical and temperate snails (Gastropoda: Littorinidae) to determine whether this group is phylogenetically constrained to freeze tolerance. We exposed dry acclimated and wet rehydrated snails to low temperatures to determine temperature of crystallisation (Tc), lower lethal temperature and LT₅₀ and to examine the relationship between ice formation and mortality. Tc was lowest in dry Afrolittorina knysnaensis (–13.6±0.4 °C), followed by dry Echinolittorina natalensis (–10.9±0.2 °C) and wet A. knysnaensis (–10.2±0.2 °C). The Tc of both A. knysnaensis and E. natalensis increased with rehydration, whereas Tc of dry and wet Afrolittorina africana did not differ (–9.6±0.2 and –9.0±0.2 °C respectively). Wet snails of all species exhibited no or low survival of inoculative freezing, whereas dry individuals of A. knysnaensis could survive subzero temperatures above –8 °C when freezing was inoculated with ice. In the absence of external ice, Afrolittorina knysnaensis employs a freeze-avoidance strategy of cold tolerance, the first time this has been reported for an intertidal snail, indicating that there is no family-level phylogenetic constraint to freeze tolerance. Echinolittorina natalensis and A. africana both showed pre-freeze mortality and survival of some internal ice formation, but were not cold hardy in any strict sense.