Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 A degrees C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 A degrees C and pH 6.5 for A. terricola, and 65 A degrees C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 A degrees C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t (50) of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4-3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and beta-xylosidase were detected which might act synergistically with xylanase.

Low Satellite DNA Variability in Natural Populations of Drosophila antonietae Involved in Different Evolutionary Events

Drosophila antonietae is a cactophilic species that is found in the mesophilic forest of the Parana`-Paraguay river basin and in the dunes of the South Atlantic coast of Brazil. Although the genetic structure of the Parana`-Paraguay river basin populations has already been established, the relationship between these populations and those on the Atlantic coast is controversial. In this study, we compared 33 repetitive units of pBuM-2 satellite DNA isolated from individuals from 8 populations of D. antonietae in these geographic regions, including some populations found within a contact zone with the closely related D. serido. The pBuM-2 sequences showed low interpopulational variability. This result was interpreted as a consequence of both gene flow among the populations and unequal crossing over promoting homogenization of the tandem arrays. The results presented here, together with those of previous studies, highlight the use of pBuM-2 for solving taxonomic conflicts within the D. buzzatii species cluster.

Microsatellite allele sequencing in population analyses of the South American cactophilic species Drosophila antonietae (Diptera: Drosophilidae)

Drosophila antonietae belongs to the Drosophila buzzatii cluster, a cactophilic group of species naturally endemic to South America. Morphological and genetic analyses indicate that its populations are the most homogenous in the cluster and that the diversity observed is mainly a result of variation within populations. Seven polymorphic microsatellite loci were described for this species and used in the present study to investigate the genetic diversity of natural populations of D. antonietae by both length and sequence variation. The study aimed to understand how homoplasy and null alleles affect inferences about the population history of this species and to obtain an accurate interpretation of population inferences where these loci could be applied. The results provide useful information on the interpretation of genetic data derived from the microsatellite loci described for D. antonietae and on evolutionary aspects of cactophilic Drosophila. Importantly, the results indicate that size homoplasy and null alleles do not represent significant problems for the population genetics analyses because the large amount of variability at microsatellite loci compensate the low frequency of these problems in the populations. (C) 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 573-584.

Re-description and new combination of five New World species of Chrysotus Meigen, with comments on the Neotropical genus Lyroneurus Loew (Diptera: Dolichopodidae)

Five New World species of Chrysotus Meigen are redescribed, four of them herein transferred from Diaphorus Meigen: C. angustifrons (Robinson), comb.nov., C. maculatus (Parent), comb.nov. (= D. maculipennis Robinson), C. robustus(Robinson), comb. nov., C. spectabilis (Loew) and C. wirthi (Robinson), comb. nov. The female of C. maculatus is described for the first time. Terminalia of both males and females are illustrated. The previous suggestion that C. angustifrons, C. robustus, C. wirthi, and C. spectabilis and the Neotropical genus Lyroneurus Loew are closely related based on wing venation similarities is analyzed and rejected.

Allozymatic divergence between border populations of two cryptic species of the Drosophila buzzatii cluster species (Diptera: Drosophilidae)

Drosophila antonietae and Drosophila gouveai are allopatric, cactophilic, cryptic and endemic of South America species, which aedeagus morphology is considered the main diagnostic character. In this work, single close populations from the edge distributions of each species, located in an ""introgressive corridor"", were analyzed regarding temporal isozenzymatic genetic variability. Isocitrate dehydrogenase (Idh) appeared as a diagnostic locus between D. antonieate and D. gouveai because each population was fixed for different alleles. Moreover, several polymorphic loci showed accentuated divergence in the allele frequency, as evidenced by Nei`s l(0.3188) and D (1.1432), and also by Reynolds` genetic distance and identity (1.3207 and 0.7331, respectively). Our results showed that, in spite of the very similar external morphology, related evolutionary histories, close distributions, and events of introgression in the studied area, these cryptic species have high allozymatic differentiation, and this is discussed here. (C) 2010 Elsevier Ltd. All rights reserved.

Spermatophore and Gonopore Morphology of the Southwestern-Atlantic Hermit Crab Pagurus exilis (Benedict, 1892) (Anomura, Paguridae)

Marcelo A. Scelzo, Marina Z. Fantucci, and Fernando L. Mantelatto (2010) Spermatophore and gonopore morphology of the southwestern-Atlantic hermit crab Pagurus exilis (Benedict, 1892) (Anomura, Paguridae). Zoological Studies 49(3): 421-433. The form and function of the spermatophore have been used as a complementary tool in studies of the reproductive biology and systematics of hermit crabs. In this context, we describe the spermatophore and gonopore morphology of Pagurus exilis. The spermatophores were extracted from the distal part of the vas deferens of specimens collected in Argentina and Brazil. The spermatophores were composed of 3 major regions: a main ampulla (with a sperm capsule inside and an accessory ampulla at the base), a stalk, and a pedestal. Each spermatophore had a distinct dorsolateral suture line around the ampulla, where the rupture occurs to release the sperm. The spermatophore total length was 1.5 times the main ampulla length. The main ampulla was oval and slightly flattened. A triangular accessory ampulla extended from the main ampulla base to the pedestal on 1 side, and contained no to several sperm. The stalk is short and flattened, and as wide as the main ampulla. One to 3 spermatophores were found attached to each pedestal, which was almost oblong in shape. The dimensions of the spermatophore and its component parts were directly influenced by the size of the hermit crab. Gonopores of males were covered by long pappose setae, while female gonopores bore a few short cuspidate setae. Specimens from Brazil and Argentina had the same spermatophore morphology, corroborating the previously observed absence of genetic differences between the both populations. The spermatophore morphology of this species has similarities with the broad general pattern of the Paguridae, being most similar to one of the (at least) 3 patterns of spermatophore morphology described for Pa gurus. http://zoolstud.sinica.edu.tw/Journals/49.3/421.pdf

Five new species of Coniceromyia Borgmeier (Diptera: Phoridae) from the Atlantic Forest, Brazil

Five new species of Coniceromyia from the Atlantic Forest in Brazil are herein described-Coniceromyia apechoneura, sp. nov., C. brandaoi, sp. nov., C. diaphaniptera, sp. nov., C. franciscana, sp. nov., and C. sanctaetheresae, sp. nov. Both C. diaphaniptera and C. franciscana have patterned wings. The male foretibia provides important diagnostic features for the species, as well as additional characters to propose clades within the genus. The male hypopygial morphology is described.

Characterization of a microcystin and detection of microcystin synthetase genes from a Brazilian isolate of Nostoc

A nostocalean nitrogen-fixing cyanobacterium isolated from an eutrophic freshwater reservoir located in Piracicaba, Sao Paulo, Brazil, was evaluated for the production of hepatotoxic cyclic heptapeptides, microcystins. Morphologically this new cyanobacterium strain appears closest to Nostoc, however, in the phylogenetic analysis of 165 rRNA gene it falls into a highly stable cluster distantly only related to the typical Nostoc cluster. Extracts of Nostoc sp. CENA88 cultured cells, investigated using ELISA assay, gave positive results and the microcystin profile revealed by ESI-Q-TOF/MS/MS analysis confirmed the production of [Dha(7)]MCYST-YR. Further, Nostoc sp. CENA88 genomic DNA was analyzed by PCR for sequences of mcyD, mcyE and mcyG genes of microcystin synthetase (mcy) cluster. The result revealed the presence of mcyD, mcyE and mcyG genes with similarities to those from mcy of Nostoc sp. strains 152 and IO-102-I and other cyanobacterial genera. The phylogenetic tree based on concatenated McyG, McyD and McyE amino acids clustered the sequences according to cyanobacterial genera, with exception of the Nostoc sp. CENA88 sequence, which was placed in a clade distantly related from other Nostoc strains, as previously observed also in the 165 rRNA phylogenetic analysis. The present study describes for the first time a Brazilian Nostoc microcystin producer and also the occurrence of demethyl MCYST-YR variant in this genus. The sequenced Nostoc genes involved in the microcystin synthesis can contribute to a better understanding of the toxigenicity and evolution of this cyanotoxin. (C) 2009 Elsevier Ltd. All rights reserved.

Developmental characterization, function and regulation of a Laccase2 encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae)

In insects, exoskeleton (cuticle) formation at each molt cycle includes complex biochemical pathways wherein the laccase enzymes (EC 1.10.3.2) may have a key role. We identified an Amlac2 gene that encodes a laccase2 in the honey bee, Apis mellifera, and investigated its function in exoskeleton differentiation. The Amlac2 gene consists of nine exons resulting in an ORE of 2193 nucleotides. The deduced translation product is a 731 amino acid protein of 81.5 kDa and a pl of 6.05. Amlac2 is highly expressed in the integument of pharate adults, and the expression precedes the onset of cuticle pigmentation and the intensification of sclerotization. In accordance with the temporal sequence of exoskeleton differentiation from anterior to posterior direction, the levels of Amlac2 transcript increase earlier in the thoracic than in the abdominal integument. The gene expression lasts even after the bees emerge from brood cells and begin activities in the nest, but declines after the transition to foraging stage, suggesting that maturation of the exoskeleton is completed at this stage. Post-transcriptional knockdown of Amlac2 gene expression resulted in structural abnormalities in the exoskeleton and drastically affected adult eclosion. By setting a ligature between the thorax and abdomen of early pupae we could delay the increase in hemolymph ecdysteroid levels in the abdomen. This severely impaired the increase in Amlac2 transcript levels and also the differentiation of the abdominal exoskeleton. Taken together, these results indicate that Amlac2 expression is controlled by ecdysteroids and has a critical role in the differentiation of the adult exoskeleton of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.

The genus Phthinia Winnertz (Diptera, Mycetophilidae) in the Neotropical region, with the first records from Brazil

Three Neotropical species of Phthinia Winnertz have been described to date. The genus is known from Chile and southern Argentina. Four new species are herein described for the genus in the region, two from Brazil-Phthinia theresae, sp. n., from the State of Espirito Santo, and Phithinia urubici, sp. n., from the State of Santa Catarina-and two from Chile-Phthinia freemani, sp. n., and Phthinia parafurcata, sp. n. Comments are made about the relationships between the Neotropical species. Some notes are added about P. furcata Freeman, P. flagellata Freeman, and P. fasciata Freeman, from Chile and southern Argentina. Attention is called for the fact that Phthinia has two species in Brazil disjunct from the other temperate species of the genus in South America, differently from most similar cases, that have a single known representative in Brazil.

Purification and Biochemical Properties of a Glucose-Stimulated beta-D-Glucosidase Produced by Humicola grisea var. thermoidea Grown on Sugarcane Bagasse

The effect of several carbon sources on the production of mycelial-bound beta-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated beta-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The beta-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50 degrees C, respectively. The purified enzyme was thermostable up to 60 min in water at 55 degrees C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, o-nitrophenyl-beta-D-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-beta-D-fucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude beta-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated beta-glucosidase from the thermophilic fungus Humicola insolens

A mycelial beta-glucosidase from the thermophilic mold Humicola insolens was purified and biochemically characterized. The enzyme showed carbohydrate content of 21% and apparent molecular mass of 94 kDa, as estimated by gel filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a single polypeptide band of 55 kDa, suggesting that the native enzyme was a homodimer. Mass spectrometry analysis showed amino acid sequence similarity with a P-glucosidase from Humicola grisea var. thermoidea, with about 22% coverage. Optima of temperature and pH were 60 degrees C and 6.0-6.5, respectively. The enzyme was stable up to I h at 50 degrees C and showed a half-life of approximately 44 min at 55 degrees C. The beta-glucosidase hydrolyzed cellobiose, lactose, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-galactopyranoside, o-nitrophenyl-beta-D-galactopyranoside, and salicin. Kinetic studies showed that p-nitrophenyl-beta-D-fucopyranoside and cellobiose were the best enzyme substrates. Enzyme activity was stimulated by glucose or xylose at concentrations up to 400 mM, with maximal stimulatory effect (about 2-fold) around 40 mM. The high catalytic efficiency for the natural substrate, good thermal stability, strong stimulation by glucose or xylose, and tolerance to elevated concentrations of these monosaccharides qualify this enzyme for application in the hydrolysis of cellulosic materials. (C) 2009 Elsevier Ltd. All rights reserved.

Organization, evolution and transcriptional profile of hexamerin genes of the parasitic wasp Nasonia vitripennis (Hymenoptera: Pteromalidae)

Hexamerins and prophenoloxidases (PPOs) proteins are members of the arthropod-haemocyanin superfamily. In contrast to haemocyanin and PPO, hexamerins do not bind oxygen, but mainly play a role as storage proteins that supply amino acids for insect metamorphosis. We identified seven genes encoding hexamerins, three encoding PPOs, and one hexamerin pseudogene in the genome of the parasitoid wasp Nasonia vitripennis. A phylogenetic analysis of hexamerins and PPOs from this wasp and related proteins from other insect orders suggests an essentially order-specific radiation of hexamerins. Temporal and spatial transcriptional profiles of N. vitripennis hexamerins suggest that they have physiological functions other than metamorphosis, which are arguably coupled with its lifestyle.

Functional and Evolutionary Insights from the Genomes of Three Parasitoid Nasonia Species

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.

The higher-level phylogeny of Archosauria (Tetrapoda: Diapsida)

Crown group Archosauria, which includes birds, dinosaurs, crocodylomorphs, and several extinct Mesozoic groups, is a primary division of the vertebrate tree of life. However, the higher-level phylogenetic relationships within Archosauria are poorly resolved and controversial, despite years of study. The phylogeny of crocodile-line archosaurs (Crurotarsi) is particularly contentious, and has been plagued by problematic taxon and character sampling. Recent discoveries and renewed focus on archosaur anatomy enable the compilation of a new dataset, which assimilates and standardizes character data pertinent to higher-level archosaur phylogeny, and is scored across the largest group of taxa yet analysed. This dataset includes 47 new characters (25% of total) and eight taxa that have yet to be included in an analysis, and total taxonomic sampling is more than twice that of any previous study. This analysis produces a well-resolved phylogeny, which recovers mostly traditional relationships within Avemetatarsalia, places Phytosauria as a basal crurotarsan clade, finds a close relationship between Aetosauria and Crocodylomorpha, and recovers a monophyletic Rauisuchia comprised of two major subclades. Support values are low, suggesting rampant homoplasy and missing data within Archosauria, but the phylogeny is highly congruent with stratigraphy. Comparison with alternative analyses identifies numerous scoring differences, but indicates that character sampling is the main source of incongruence. The phylogeny implies major missing lineages in the Early Triassic and may support a Carnian-Norian extinction event.