Species specificity and intraspecific variation in the chemical profiles of Heliconius butterflies across a large geographic range

In many animals, mate choice is important for the maintenance of reproductive isolation between species. Traits important for mate choice and behavioral isolation are predicted to be under strong stabilizing selection within species; however, such traits can also exhibit variation at the population level driven by neutral and adaptive evolutionary processes. Here, we describe patterns of divergence among androconial and genital chemical profiles at inter‐ and intraspecific levels in mimetic Heliconius butterflies. Most variation in chemical bouquets was found between species, but there were also quantitative differences at the population level. We found a strong correlation between interspecific chemical and genetic divergence, but this correlation varied in intraspecific comparisons. We identified “indicator” compounds characteristic of particular species that included compounds already known to elicit a behavioral response, suggesting an approach for identification of candidate compounds for future behavioral studies in novel systems. Overall, the strong signal of species identity suggests a role for these compounds in species recognition, but with additional potentially neutral variation at the population level.     

Contribution of single tryptophan residues to the fluorescence and stability of ribonuclease Sa

Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.     

A Simple Geometrical Pattern for the Branching Distribution of the Bronchial Tree, Useful to Estimate Optimality Departures

The design of the bronchial tree has largely been proposed as a model of optimal design from a physical-functional perspective. However, the distributive function of the airway may be more related to a geometrical than a physical problem. The bronchial tree must distribute a three dimensional volume of inspired air on a two dimensional alveolar surface, included in a limited volume. It is thus valid to ask whether an optimal bronchial tree from a physical perspective is also optimum from a geometrical point of view. In this paper we generate a simple geometric model for the branching pattern of the bronchial tree, deducing relationships that permit estimation of the departures from the geometrical optimum of each bifurcation. We also, for comparative purposes, estimate the departures from the physical optimum. From the geometrical assumptions: i) a symmetrical dichotomic fractal design, ii) with minimum volume and iii) maximum dispersion of the terminal points; and several simulations we suggest that the optimality is characterized by a bifurcation angle theta approximately 60 degrees and a length reduction scale gamma = (1/2)(1/3) = 0.7937. We propose distances from the physical and geometrical optimality defined as Euclidean distances from the expected optima. We show how the advanced relationships and the distances can be used to estimate departures from the optimality in bronchographs of four species. We found lower physical and geometrical departures in the distal zone than those of the proximal zones, as well as lower physical than geometrical departures from optimality.     

Diet of Southern Muriquis in Continuous Brazilian Atlantic Forest

We systematically collected data on feeding behavior for one group of 33–39 southern muriquis (Brachyteles arachnoides) in Parque Estadual Carlos Botelho (PECB), São Paulo State, Brazil (37,432.45 ha of continuous Atlantic Forest), between January and December 1995. We determined food item consumption from instantaneous scans of behavior. Fruits were the most eaten food items in all 12 mo (40–80% of scan in every mo, average = 71.3%). Muriquis ate young leaves more than mature leaves or flowers. Our results are consistent with previous findings at the same and neighboring forest sites that southern muriquis have a consistently frugivorous diet when inhabiting less disturbed habitats, but contrast with previous observations on oppportunistic frugivory in muriqui populations inhabiting fragmented forests. Sustained high levels of frugivory probably result from year-round availability of fruit within large continuous forests.     

A hybrid zone provides evidence for incipient ecological speciation in Heliconius butterflies

In Heliconius butterflies, it has been proposed that speciation occurs through a combination of divergence in ecological habitat preferences and mimetic colour patterns. Here we test this hypothesis by investigating a parapatric form of the widespread species Heliconius erato. Mendelian (colour patterns) and molecular genetic data permit us to address hypotheses about introgression and genetic differentiation between different populations. Combined analysis of colour pattern, microsatellite loci and mitochondrial DNA showed that Heliconius erato venus and Heliconius erato chestertonii form a bimodal hybrid zone implying partial reproductive isolation. In a sample of 121 individuals collected in sympatry, 25% were hybrids representing a significant deficit of heterozygotes compared to the Hardy-Weinberg expectation. Seven microsatellite loci, analysed for a subset of these individuals, showed marked differentiation between the parental taxa, and unambiguously identified two genotypic clusters concordant with our phenotypic classification of individuals. Mitochondrial DNA analysis showed H. erato venus as a monophyletic group well differentiated from H. erato chestertonii, implying a lack of historical introgression between the populations. Heliconius erato chestertonii is therefore an incipient species that maintains its integrity despite high levels of hybridization. Moreover, H. erato chestertonii is found at higher altitudes than other races of H. erato and has a distinct colour pattern and mimetic relationship. Hence, there are now two examples of parapatric incipient species related to H. erato, H. himera and H. erato chestertonii, both of which are associated with higher altitudes, more arid habitats and distinct mimetic relationships. This implies that parapatric habitat adaptation is a likely cause of speciation in this group.     

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS‐PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29‐kDa band from the cell‐free culture medium was clearly observed by SDS‐PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 μg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

New unsymmetric dinuclear Cu(II)Cu(II) complexes and their relevance to copper(II) containing metalloenzymes and DNA cleavage

The new homodinuclear complexes, [Cu(2)(II)(HLdtb)(mu-OCH(3))](ClO(4))(2) (1) and [Cu(2)(II)(Ldtb)(mu-OCH(3))](BPh(4)) (2), with the unsymmetrical N(5)O(2) donor ligand (H(2)Ldtb) - {2-[N,N-Bis(2-pyridylmethyl)aminomethyl]-6-[N',N'-(3,5-di-tert-butylbenzyl-2-hydroxy)(2-pyridylmethyl)]aminomethyl}-4-methylphenol have been synthesized and characterized in the solid state by X-ray crystallography.In both cases the structure reveals that the complexes have a common {Cu(II)(mu-phenoxo)(mu-OCH(3))Cu(II)} structural unit.Magnetic susceptibility studies of 1 and 2 reveal J values of -38.3 cm(-1) and -2.02 cm(-1), respectively, and that the degree of antiferromagnetic coupling is strongly dependent on the coordination geometries of the copper centers within the dinuclear {Cu(II)(mu-OCH(3))(mu-phenolate)Cu(II)} structural unit.Solution studies in dichloromethane, using UV-Visible spectroscopy and electrochemistry, indicate that under these experimental conditions the first coordination spheres of the Cu(II) centers are maintained as observed in the solid state structures, and that both forms can be brought into equilibrium ([Cu(2)(HLdtb)(mu-OCH(3))](2+)=[Cu(2)(Ldtb)(mu-OCH(3))](+)+H(+)) by adjusting the pH with Et(3)N (Ldtb(2-) is the deprotonated form of the ligand).On the other hand, potentiometric titration studies of 1 in an ethanol/water mixture (70:30 V/V; I=0.1M KCl) show three titrable protons, indicating the dissociation of the bridging CH(3)O(-) group.The catecholase activity of 1 and 2 in methanol/water buffer (30:1 V/V) demonstrates that the deprotonated form is the active species in the oxidation of 3,5-di-tert-butylcatechol and that the reaction follows Michaelis-Menten behavior with k(cat)=5.33 x 10(-3)s(-1) and K(M)=3.96 x 10(-3)M. Interestingly, 2 can be electrochemically oxidized with E(1/2)=0.27 V vs.Fc(+)/Fc (Fc(+)/Fc is the redox pair ferrocinium/ferrocene), a redox potential which is believed to be related to the formation of a phenoxyl radical.Since these complexes are redox active species, we analyzed their activity toward the nucleic acid DNA, a macromolecule prone to oxidative damage.Interestingly these complexes promoted DNA cleavage following an oxygen dependent pathway.     

Determination of the refractive index increment (dn/dc) of molecule and macromolecule solutions by surface plasmon resonance

An automated method for dn/dc determination using a surface plasmon resonance instrument in tandem with a flow injection gradient system (FIG-SPR) is proposed. dn/dc determinations of small molecule and biomolecule, surfactant, polymer, and biopolymer solutions with precision around 1-2% and good accuracy were performed using the new method. dn/dc measurements were also carried out manually on a conventional SPR equipment with similar accuracy and precision. The FIG-SPR instrument is inexpensive and could be easily coupled to commercially available SPR and liquid chromatography instruments to obtain several properties of the solutions, which are based on measurements of refractive index.     

Synthesis, characterization and photophysical properties of mixed ligand tris(polypyridyl)chromium(III) complexes, [Cr(phen)2L]

A series of new heteroleptic, tris(polypyridyl)chromium(III) complexes, [Cr(phen)₂L]³⁺ (L = substituted phenanthrolines or bipyridines), has been prepared and characterized, and their photophyical properties in a number of solvents have been investigated. X-ray crystallography measurements confirmed that the cationic (3+) units contain only one ligand L plus two phenanthroline ligands. Electrochemical and photophysical data showed that both ground state potentials and lifetime decays are sensitive to ligand structure and the nature of the solvent with the exception of compounds containing L = 5-amino-1,10-phenanthroline (aphen) and 2,2′-bipyrimidine (bpm). Addition of electron-donating groups in the ligand structure shifts redox potentials to more negative values than those observed for the parent compound, [Cr(phen)₃]³⁺. Emission decays show a complex dependence with the solvent. The longest lifetime was observed for [Cr(phen)₂(dip)]³⁺ (dip = 4,7-diphenylphenanthroline) in air-free aqueous solutions, τ = 273 μs. Solvent effects are explained in terms of the affinity of hydrophobic complexes for non-polar solvent molecules and the solvent microstructure surrounding chromium units.