A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis

Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species‐specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real‐time high‐resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species‐specific genetic mutations that result in PCR products with unique melt profiles. A real‐time HRM PCR species‐diagnostic assay (RT‐HRM‐PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing.     

适应不同光环境小蜡叶片气孔导度、气孔开度和气孔密度的时空变化

对生长在强光环境和弱光环境小蜡叶片的气孔参数测定发现:气孔导度和气孔开度在4个取样部位存在异质性分布.气孔导度和气孔开度经回归分析呈线性、指数或多项式分布.统计分析表明:强光下的叶片气孔导度和气孔开度的相关性明显高于弱光环境叶片的数值.无论强光环境还是弱光环境下的叶片,在取样部位编号为1和3,其气孔导度和气孔开度均存在显著的线性关系.弱光环境下叶片的气孔密度要远低于强光环境下的叶片.强光环境下叶片对变化环境的敏感性要大于弱光环境下的叶片,这可能与强光环境叶片具有较高的气孔密度有关.     

适应不同光环境小蜡叶片气孔导度、气孔开度和气孔密度的时空变化

对生长在强光环境和弱光环境小蜡叶片的气孔参数测定发现 :气孔导度和气孔开度在 4个取样部位存在异质性分布。气孔导度和气孔开度经回归分析呈线性、指数或多项式分布。统计分析表明 :强光下的叶片气孔导度和气孔开度的相关性明显高于弱光环境叶片的数值。无论强光环境还是弱光环境下的叶片 ,在取样部位编号为1和 3,其气孔导度和气孔开度均存在显著的线性关系。弱光环境下叶片的气孔密度要远低于强光环境下的叶片。强光环境下叶片对变化环境的敏感性要大于弱光环境下的叶片 ,这可能与强光环境叶片具有较高的气孔密度有关。     

Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves

Sugar content and freezing tolerance of protoplasts of Arabidopsis thaliana leaves were manipulated by incubating seedlings in a sucrose solution before protoplast isolation. Incubation in a 400 mM sucrose solution at 2 °C in the dark increased their freezing tolerance equivalent to that achieved after a conventional cold acclimation at 2 °C. The increased freezing tolerance was due to a decrease in the incidence of freeze‐induced lesions: expansion‐induced lysis (EIL) between ?2 and ?4 °C and loss of osmotic responsiveness (LOR) between ?5 and ?12 °C. The concentration of sucrose in the incubation medium required to minimize the incidence of the lesions was substantially different: 10–35 mM for EIL and 30–400 mM for LOR. Incubation in the sucrose solution at 23 °C decreased LOR only at ?5 and ?6 °C but less than that incubated at 2 °C, and there was no effect on EIL. Incubation in sorbitol solutions at 2 °C also decreased LOR at ?5 and ?6 °C but much less than in the sucrose solution. These results suggest that low concentrations of sucrose act as a metabolic substrate for the low‐temperature‐induced alterations required for the amelioration of EIL and, at higher concentrations, sucrose has a direct cryoprotective effect to minimize LOR.     

Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15°C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37°C membranes, while 15°C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15°C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15°C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37°C, but only 50% at 15°C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15°C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Polymorphic microsatellite markers for studies of the conservation and reproductive genetics of imperilled sand tiger sharks (Carcharias taurus)

We report on the isolation of eight microsatellites from the sand tiger shark, Carcharias taurus, using an enrichment protocol. All loci, with the exception of Cta45–183, were in Hardy–Weinberg equilibrium. Loci exhibited three to 15 alleles, and observed and expected heterozygosities of 0.095–1.000 and 0.284–0.924, respectively. An additional marker (Iox‐12) developed from a shortfin mako library was variable in sand tigers. These markers will be used to examine population genetics and mating patterns of this imperilled species.     

Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation

Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2 degrees C for as short as 1 day, and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO2 fixation, and proteolysis.     

Protoplast rotation in a rotating electric field: The influence of cold acclimation

Summary An experimental and theoretical investigation has been made of the rotation of protoplasts ofSecale cereale L. (cv Puma) in a rotating electric field for the purpose of determining the electrical properties of the protoplast plasma membrane. The dependence of the protoplast rotation rate on: (1) the rotation rate of the applied electric field; (2) the electrical conductivity of the external medium; and (3) cold acclimation or lack thereof were determined. A theoretical analysis of the rotation rate of polarizable spherical cells in a rotating electric field leads to a qualitatively similar formula to that of Arnold and Zimmermann (Z. Naturforsch. 37:908–915, 1982), but it differs from this earlier work by a large numerical factor (180). Detailed comparisons of the observed protoplast rotation rates with the new theory show generally good agreement. The protoplast rotation measurements allow a noninvasive determination of the specific plasma membrane capacitance,c _m. The average value found in the present experiments isc _m=(0.56±0.08)×10^–2 F/m². Within the experimental errors, thec _m values are the same for cold-acclimated and noncold-acclimated protoplasts. Determination of plasma membrane resistance from protoplast rotation measurements does not appear feasible because of the high values of the specific resistance.     

A study on the dynamic features of photosystem stoichiometry: Accomplishments and problems for future studies

Our study on the regulation by light of photosystem stoichiometry in cyanophytes is briefly reviewed here. It can be summarized as follows: Adjustment of photosystem stoichiometry results in optimization of photosynthetic efficiency under the prevailing light-growth conditions. Photosystem (PS) I is primarily affected, increasing or decreasing, relative to PS II. The regulation of PS I synthesis appears to be at the translation or the post-translational level. The regulation is probably governed by changes in the redox level of the electron transport components, most probably at Cyt b₆in the Cyt b₆–f complex. Our results to date are at the physiological level, but they have raised many questions and provided suggestions for future directions in exploring the mechanism of regulation, some of which are discussed in this perspective.     

Red muscle function in stiff-bodied swimmers: there and almost back again

Fishes with internalized and endothermic red muscles (i.e. tunas and lamnid sharks) are known for a stiff-bodied form of undulatory swimming, based on unique muscle-tendon architecture that limits lateral undulation to the tail region even though the red muscle is shifted anteriorly. A strong convergence between lamnid sharks and tunas in these features suggests that thunniform swimming might be evolutionarily tied to this specialization of red muscle, but recent observations on the common thresher shark (Alopias vulpinus) do not support this view. Here, we review the fundamental features of the locomotor systems in lamnids and tunas, and present data on in vivo muscle function and swimming mechanics in thresher sharks. These results suggest that the presence of endothermic and internalized red muscles alone in a fish does not predict or constrain the swimming mode to be thunniform and, indeed, that the benefits of this type of muscle may vary greatly as a consequence of body size.     

Observations on the Oman Shark,Iago omanensis (Triakidae), with emphasis on the morphological and cytological changes of the oviduct and yolk sac during gestation

The shark Iago omanensis (Triakidae, Selachia) is encountered in large populations in the Gulf of Aqaba, Red Sea, at depths of 150–1,500 m. It is a placental viviparous species, reproductive all year round and giving birth to four (occasionally five) young of 170- to 180-mm total length (TL). Its distribution and morphometrics, as well as histological and cytological changes in the oviducts, were studied. The ratio of weight of the female genital organs to body weight changes from 0.7% in nongravid females to 19.8% in the final stages of pregnancy. The ripe, liberated eggs, which are 11–12 mm long and 5 mm wide, pass through the nidamental gland and settle in the uterus. The embryo attains 9- to 11-mm TL and settles on a protruding ridge of the submucosa, covered with a microvillar endometrium. At this site of attachment, a placenta is formed and the participating uterine endometrium and wall of the yolk sac undergo profound histocytological changes, forming two parts of this organ. Three forms of food provisioning occur in the growing embryos: (1) lecithotrophic, based on yolk transported from the egg to the embryonic gut via the umbilical cord; (2) mixed food provision, during which, in addition to nourishment provided via the umbilicus, food is transported across the placenta through transfer from the female blood vascular system to the embryonic yolk sac via the trophic villi of the yolk sac; and (3) histotrophic, when all yolk reserves have been used and nutrition is provided from the so-called “milk” within the yolk sac, metabolized by the trophic structures of the sac and transported by blood vessels. Despite the gradual utilization of yolk, the yolk sac mass initially increases from 0.5–1.0 cc to 2.0–2.2 cc with the addition of primary and secondary trophic villi until, during the final stages of embryogenesis, it decreases again to 1.4–1.6 cc. Neonate juveniles are 35–40 times heavier than the original eggs. J. Morphol. 236:151–165, 1998. © 1998 Wiley-Liss, Inc.