Temperature coupling in cricket acoustic communication

Acoustic communication in Gryllus firmus is temperature-coupled: temperature induces parallel changes in male calling song temporal pattern, and in female preference for song. Temperature effects on song production and recognition networks were localized by selectively warming head or thorax or both head and thorax of intact crickets, then eliciting aggression song production (males) or phonotaxis to synthetic calling song (females). Because male song is produced by a thoracic central pattern generator (CPG), and because head ganglia are necessary for female song recognition, measurements of female phonotaxis under such conditions may be used to test the following competing hypotheses about organization of the song recognition network: 1. A set of neurons homologous to the male song CPG exist in the female, and are used as a template that determines preferred values of song temporal parameters for song pattern recognition (the common neural elements hypothesis), and 2. temporal pattern preference is determined entirely within the head ganglia. Neither selective warming of the head nor of the thorax was effective in changing female song preference, but simultaneous warming of head and thorax shifted preference toward a faster song in most preparations, as did warming the whole animal by raising ambient temperature. These results suggest that phonotactic preference for song temporal pattern is plurisegmentally determined in field crickets. Selective warming experiments during aggression song production in males revealed that syllable period is influenced but not completely determined by thoracic temperature; head temperature is irrelevant. The song CPG appears to receive some rate-setting information from outside the thoracic central nervous system.     

Pleistocene megafaunal extinctions and the functional loss of long‐distance seed‐dispersal services

Pleistocene extinctions affected mainly large‐bodied animals, determining the loss or changes in numerous ecological functions. Evidence points to a central role of many extinct megafauna herbivores as seed dispersers. An important step in understanding the legacy of extinct mutualistic interactions is to evaluate the roles and effectiveness of megafauna herbivores in seed dispersal. Here we use morphological and ecophysiological allometries to estimate both quantitative and qualitative aspects of seed‐dispersal services likely provided by extinct megafauna. We developed a mechanistic model that encompasses four stages of seed dispersal – seed ingestion, gut retention, animal movement, and seed deposition. We estimate seed‐dispersal kernels through simulations to infer the role of Pleistocene megafauna in promoting long‐distance dispersal and examine how seed dispersal was affected by extinctions. Simulations suggest extinct large‐bodied frugivores would frequently disperse large seeds over a thousand meters, whereas smaller‐bodied frugivores are more likely to deposit the seeds over a few hundred meters. Moreover, events of long‐distance seed dispersal by the extinct megafauna would be up to ten times longer than long‐distance dispersal by smaller‐sized extant mammals. By estimating the combined distribution of seed dispersal distances considering all large‐bodied mammalian frugivores in specific South American Pleistocene assemblages we found that long‐distance dispersal contracted by at least two thirds after the megafauna died out. The disruption of long‐distance dispersal is expected to have consequences for recruitment, spatial and genetic structure of plant populations, population persistence and community composition. Promoting long‐distance seed dispersal was one among other salient features of extinct Pleistocene megafauna that reveal their influence on natural ecosystems. Modeling the consequences of megafaunal extinctions can offer quantitative predictions on the consequences of ongoing defaunation to plant populations and ecological communities.     

Cloning,expression, purification and spectrophotometric analysis of lanosterol 14-alpha demethylase from <Emphasis Type="Italic">Leishmania braziliensis</Emphasis> (<Emphasis Type="Italic">Lb</Emphasis>CYP51)

Leishmaniasis, a neglected tropical disease, is a major cause of morbidity and mortality worldwide. Of the three main clinical forms, cutaneous leishmaniasis (CL) is the most common and 40 million people are at risk in the endemic areas. Currently, the available drugs to fight leishmaniasis have high toxicity and poor efficiency. Then, it is very important to search for effective and safe drugs that would target essential enzymes from the parasite, such as lanosterol 14-alpha demethylase (CYP51, EC 1.14.13.70) from Leishmania braziliensis. Because most drug design efforts have been directed for Leishmania non-braziliensis species, there is no structural or kinetic data regarding L. braziliensis CYP51. Herein, we present for the first time molecular biology efforts and purification protocol to obtain the enzyme LbCYP51. These results lay the ground for future investigation of drugs against this target.     

Evaluation of Biolog system for identification of strains of Paenibacillus azotofixans

Biolog system was evaluated for the identification of strains of Paenibacillus azotofixans as no data concerning this species were in the list of Bacillus currently identified using Biolog data base. The P. azotofixans type strain P3L5 was first tested with the results recorded manually or using the automatic plate reader. In both cases, P3L5 utilized 22 carbon sources and when the results obtained were compared to data of Biolog software (Release 3.50), P3L5 was identified as B. azotoformans with a similarity coefficient of 0.913 (data recorded manually) and of 0.791 (data recorded automatically). Metabolic profiles of P3L5 were also compared after readings of 4 and 24 h using the computer-driven automatic plate reader. No significant difference was observed in both cases and P3L5 was identified again as B. azotoformans with indices of similarity considered only for excellent identification. Besides P3L5, other 15 P. azotofixans strains were tested with the Biolog system and all were identified as B. azotoformans with similarity coefficients varying from 0.511 to 0.927. Phenotypic and genetic characteristics of B. azotoformans were compared to those described for P. azotofixans to explain the misidentification of the latter species. We could conclude that these two species are quite different and that data of Biolog software are from P. azotofixans and not from B. azotoformans.     

Cardosin A, an abundant aspartic proteinase, accumulates in protein storage vacuoles in the stigmatic papillae of Cynara cardunculus L.

The function of aspartic proteinases (EC 3.4.23) present in flowers of Cynara species is still unknown. Cardosin A, as a highly abundant aspartic proteinase from Cynara cardunculus L., a relative of the artichoke, is synthesised as a zymogen and subsequently undergoes proteolytic processing, yielding the mature and active enzyme. Here we report the study of the expression and localization of cardosin A, as a first approach to address the question of its physiological relevance. A polyclonal antibody specific for cardosin A was raised against a synthetic peptide corresponding to an amino acid sequence of the enzyme. This antibody was used to study the organ-specific, tissue-specific and subcellular localization of cardosin A by immunoblotting, tissue printing and immunogold electron microscopy. The results showed that expression of cardosin A is highly restricted to the pistils, and that the enzyme accumulates mainly in protein storage vacuoles of the stigmatic papillae. Cardosin A is also present, although much less abundantly, in the vacuoles of the cells of the epidermis of the style. In view of these results, the possible physiological roles of cardosin A are discussed, namely an involvement in defense mechanisms or pollen-pistil interaction, as well as in flower senescence. Received: 10 December 1996 / Accepted: 14 March 1997     

Partial purification and some properties of shikimate dehydrogenase from tomatoes

The partial purification of shikimate dehydrogenase (SDH) from tomato fruit was achieved by precipitation with ammonium sulphate, and chromatography on DEAE-cellulose and hydroxyapatite. The enzyme has a MW of 73000, shows an optimum at pH 9.1 and K_m values of 3.8 × 10^?5 M and 1.0 × 10^?5 M with shikimic acid and NADP as substrates. NADP could not be replaced by NAD. The tomato enzyme is competitively inhibited by protocatechuic acid with a K_i value of 7.7 × 10^?5 M. On the other hand, cinnamic acid derivatives and 2-hydroxybenzoic acid were ineffective. At 50° for 5 min the SDH is inactivated by 85%. The activity was inhibited by pCMB and N-ethylmaleimide, suggesting a requirement for SH groups. The inactivation plot of oxidation by pCMB was biphasic, and NADP decreased the reactivity of sulphydryl groups to the reagent. The activation energy was found to be 14.2kcal/mol. The properties of the SDH are discussed in relation to the enzymes from other sources.     

Shifts in the abundance and distribution of shallow water fish fauna on the southeastern Brazilian coast: a response to climate change

Myriophyllum aquaticum is a semi-submerged exotic macrophyte that was introduced to China for many years. This species may be found in an emergent form in aquatic environments or in an amphibious form under drained conditions. Nuisance growth of this species has often been attributed to excessive amounts of nutrients. Therefore, we tested the following hypotheses: (1) high nutrient availability facilitates the establishment of M. aquaticum and (2) fragment type interacts with nutrient availability to determine the colonization and regeneration capacities of M. aquaticum. Two types of fragments were grown in water solutions with two levels of phosphorous. After 3 weeks, the survival rates showed no significant difference between the phosphorous treatments. However, emergent fragments showed higher RGR in the low and high phosphorous treatments than amphibious fragments. In addition, emergent fragments also showed higher regeneration capacities, indicating higher invasiveness in emergent fragments compared to amphibious fragments. Moreover, the high phosphorous concentration caused emergent fragments to produce more branches, indicating that nutrient availability may increase M. aquaticum propagule pressure. Our study highlights that nutrient supply increased emergent fragment establishment and shaped the invasion dynamics of macrophytes, which could help predict the spread and potential impact of exotic macrophytes in natural aquatic ecosystems.     

Diversification by host switching and dispersal shaped the diversity and distribution of avian malaria parasites in Amazonia

Understanding how pathogens and parasites diversify through time and space is fundamental to predicting emerging infectious diseases. Here, we use biogeographic, coevolutionary and phylogenetic analyses to describe the origin, diversity, and distribution of avian malaria parasites in the most diverse avifauna on Earth. We first performed phylogenetic analyses using the mitochondrial cytochrome b (cyt b) gene to determine relationships among parasite lineages. Then, we estimated divergence times and reconstructed ancestral areas to uncover how landscape evolution has shaped the diversification of Parahaemoproteus and Plasmodium in Amazonia. Finally, we assessed the coevolutionary patterns of diversification in this host–parasite system to determine how coevolution may have influenced the contemporary diversity of avian malaria parasites and their distribution among Amazonian birds. Biogeographic analysis of 324 haemosporidian parasite lineages recovered from 4178 individual birds provided strong evidence that these parasites readily disperse across major Amazonian rivers and this has occurred with increasing frequency over the last five million years. We also recovered many duplication events within areas of endemism in Amazonia. Cophylogenetic analyses of these blood parasites and their avian hosts support a diversification history dominated by host switching. The ability of avian malaria parasites to disperse geographically and shift among avian hosts has played a major role in their radiation and has shaped the current distribution and diversity of these parasites across Amazonia.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection.

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes ('lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin N-terminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.