Assessment of different sperm functional tests in golden‐headed lion tamarins (Leontopithecus chrysomelas)

The golden‐headed lion tamarin (Leontopithecus chrysomelas) is an endangered species endemic to Brazil's Atlantic Forest, a shrinking biodiversity hotspot. As in other Neotropical primates, its semen characteristics and freezability are poorly studied. Hence, reproductive technologies for callitrichids would greatly benefit from reliable methods of semen analysis. In a bid to promote reproductive research in tamarins, we validated simple and inexpensive sperm function tests that can be used to monitor sperm‐egg binding, plasma membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. Ejaculates from adult males were individually diluted and divided into control and damage‐induced aliquots, and then samples comprising assorted amounts of damaged spermatozoa were examined by organelle‐specific tests. Our findings showed that sperm‐binding in chicken egg perivitelline membrane (EPM) positively correlated with the number of spermatozoa injured by snap‐freezing. Eosin‐nigrosin (EN) and propidium iodide readings were correlated with each other, and both provided robust measurements of plasma membrane integrity. A high correlation between expected and measured amounts of acrosome‐intact spermatozoa was found using Fast Green‐Rose Bengal (FG‐RB), Coomassie Blue (CB), and FITC‐PSA stains, and all three methods exhibited comparable results. Likewise, different percentages of UV‐irradiated spermatozoa were accurately assessed for DNA integrity by Toluidine Blue (TB) and sperm chromatin dispersion (SCD) tests. Comparisons between 3,3′‐diaminobenzidine (DAB) and JC‐1 stains also indicated the reliability of the former assay to ascertain gradual increases in spermatozoa with greater mitochondrial function. These data confirmed that different parts of the tamarin spermatozoa can be simply and consistently evaluated by EPM, EN, FG‐RB, CB, TB, and DAB protocols.     

DNA Barcoding of Neotropical Sand Flies (Diptera,Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.     

Hydrogen turnover and subcellular compartmentation of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate as detected by (13)C NMR.

(13)C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate with deuterons from intracellular heavy water providing information on alpha-ketoglutarate/glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-(13)C]alanine in buffer containing or not 50% (2)H(2)O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution (13)C NMR (150.13 MHz) with (1)H decoupling only and with simultaneous (1)H and (2)H decoupling. (13)C-(2)H couplings and (2)H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; min(-1)) for (i) the reversible exchange of [2-(13)C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-(13)C]glutamate H3(proS) as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3(proR) and H3(proS) as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of (1)H-(2)H exchange for the H2 (0.4, 0.5) or H3(proR) (0.3, 0.2) or the H2 and H3(proS) hydrogens (0.20, 0.23) of [3-(13)C]aspartate isotopomers. The ubiquitous subcellular localization of (1)H-(2)H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-(13)C]glutamate and [3-(13)C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria.     

Sequence of candicidin action on yeast célls

The action of the polyene antibiotic candicidin (a heptaene) on yeast cells in growth medium has been studied. Candicidin at the growth-inhibiting concentration produced the following sequence of events. (1) There was an immediate and rapid loss of K⁺. (2) After about 10 min, Mg²⁺ was lost to the medium and protein synthesis and uptake of amino acids decreased rapidly while the intracellular ATP level rose. Candicidin caused a temporary stimulation of amino acid uptake in salt-free buffer. (3) After about 20 min RNA synthesis and glucose consumption declined. Transport of sugars did not appear to be inhibited but energy-dependent accumulation and assimilation were sharply restricted. (4) Candicidin did not cause release of phosphate, amino acids, uracil or uridine from the internal pools, or make the membrane permeable to uridine 5′-phosphate or ATP. Damage to the cell membrane by candicidin appears to be relatively slight and affects primarily membrane components related to ion transport. The various metabolic changes noted probably result from the K⁺ loss and the eventual decline of the energy-generating systems.     

Temperature influences habitat preference of coral reef fishes: Will generalists become more specialised in a warming ocean?

Climate change is expected to pose a significant risk to species that exhibit strong behavioural preferences for specific habitat types, with generalist species assumed to be less vulnerable. In this study, we conducted habitat choice experiments to determine how water temperature influences habitat preference for three common species of coral reef damselfish (Pomacentridae) that differ in their levels of habitat specialisation. The lemon damselfish Pomacentrus moluccensis, a habitat specialist, consistently selected complex coral habitat across all temperature treatments (selected based on local average seasonal temperatures naturally experienced in situ: ambient winter 22°C; ambient summer 28°C; and elevated 31°C). Unexpectedly, the neon damselfish Pomacentrus coelestis and scissortail sergeant Abudefduf sexfasciatus, both of which have more generalist habitat associations, developed strong habitat preferences (for complex coral and boulder habitat, respectively) at the elevated temperature treatment (31°C) compared to no single preferred habitat at 22°C or 28°C. The observed shifts in habitat preference with temperature suggest that we may be currently underestimating the vulnerability of some habitat generalists to climate change and highlight that the ongoing loss of complex live coral through coral bleaching could further exacerbate resource overlap and species competition in ways not currently considered in climate change models.     

Ecophysiological factors affecting growth,sporulation and survival of the biocontrol agent Penicillium oxalicum

The effect of temperature, pH, water potential and sources of nitrogen and carbon on the biocontrol agent Penicillium oxalicum were studied in vitro. The fungus is xerotolerant, mesophillic and has a wide pH tolerance. The parameters evaluated (germination, germ tube length, growth rate and sporulation) showed different sensitivities to the environmental factors. Peptone and free amino acids gave the highest growth rates and high levels of sporulation. Xylose, mannose and fructose gave the highest growth rates and mannose induced strong sporulation. The effect of nutrients (mannose + arginine) and water potential was also studied in vivo. The xerotolerant character of the fungus was confirmed. From this study we consider Penicillium oxalicum ecologically competent to perform effectively as a biocontrol agent in the soil environment. This revised version was published online in June 2006 with corrections to the Cover Date.