Bacterial Community Associated with Healthy and Diseased Reef Coral Mussismilia hispida from Eastern Brazil

In order to characterize the bacterial community diversity associated to mucus of the coral Mussismilia hispida, four 16S rDNA libraries were constructed and 400 clones from each library were analyzed from two healthy colonies, one diseased colony and the surrounding water. Nine bacterial phyla were identified in healthy M. hispida, with a dominance of Proteobacteria, Actinobacteria, Acidobacteria, Lentisphaerae, and Nitrospira. The most commonly found species were related to the genera Azospirillum, Hirschia, Fabibacter, Blastochloris, Stella, Vibrio, Flavobacterium, Ochrobactrum, Terasakiella, Alkalibacter, Staphylococcus, Azospirillum, Propionibacterium, Arcobacter, and Paenibacillus. In contrast, diseased M. hispida had a predominance of one single species of Bacteroidetes, corresponding to more than 70% of the sequences. Rarefaction curves using evolutionary distance of 1% showed a greater decrease in bacterial diversity in the diseased M. hispida, with a reduction of almost 85% in OTUs in comparison to healthy colonies. ∫-Libshuff analyses show that significant p values obtained were <0.0001, demonstrating that the four libraries are significantly different. Furthermore, the sympatric corals M. hispida and Mussismilia braziliensis appear to have different bacterial community compositions according to Principal Component Analysis and Lineage-specific Analysis. Moreover, lineages that contribute to those differences were identified as α-Proteobacteria, Bacteroidetes, and Firmicutes. The results obtained in this study suggest host–microbe co-evolution in Mussismilia, and it was the first study on the diversity of the microbiota of the endemic and endangered of extinction Brazilian coral M. hispida from Abrolhos bank.     

Molecular tracking of jaguar melanism using faecal DNA

Major evolutionary questions remain elusive due to persistent difficulties in directly studying the genetics of variable phenotypes in natural populations. Many phenotypic variants may be of adaptive relevance, and thus important to consider in the context of conservation genetics. However, since the dynamics of these traits is usually poorly understood in the wild, their incorporation in conservation strategies is difficult to accomplish. For animals which exhibit intriguing phenotypic variation but are difficult to track in the wild, innovative approaches are required to investigate such issues. Here we demonstrate that non-invasive DNA sampling can be used to study the genetics and ecology of melanism in the jaguar, by directly genotyping the molecular polymorphism underlying this coloration trait. These results open new prospects for the in-depth investigation of this polymorphism, and highlight the broader potential of non-invasive DNA-based phenotype tracking for wildlife in general.     

The acceptance rate of young wasps by alien colonies depends on colony developmental stages in the swarm-founding wasp, Polybia paulista von ihering (Hymenoptera: Vespidae)

In social insects, newly emerged individuals learn the colony-specific chemical label from their natal comb shortly after their emergence. These labels help to identify each individual's colony of origin and are used as a recognition template against which individuals can discriminate nestmates from non-nestmates. Our previous studies with Polybia paulista von Ihering support this general pattern, and the acceptance rate of young female and male wasps decreased as a function of their age. Our study also showed in P. paulista that more than 90% of newly emerged female wasps might be accepted by conspecific unrelated colonies. However, it has not been investigated whether the acceptance rate of newly emerged female wasps depends on colony developmental stage of recipient colonies. We introduced newly emerged female wasps of P. paulista into different colony developmental stags of recipient colonies, i.e., worker-producing and male-producing colonies. We found that the acceptance rate of newly emerged female wasps by alien colonies was pretty lower by male-producing colonies than worker-producing colonies. This is the first study to show that the acceptance rate of young female wasps depends on stages of recipient colonies.     

Factors affecting conception rate after artificial insemination and pregnancy loss in lactating dairy cows

Objectives were to determine factors associated with conception rate (CR) and pregnancy loss (PL) in high producing lactating Holstein cows. In Study 1, CR was evaluated in 7633 artificial inseminations (AI) of 3161 dairy cows in two dairy farms. Pregnancy diagnosis was performed by palpation per rectum 39+/-3 days after AI. Environmental temperature was recorded at different intervals prior to and after AI. In Study 2, 1465 pregnancies from 1393 cows diagnosed at 31+/-3 days after AI by ultrasonography on three dairy farms were re-examined 14 days later to determine PL. Temperature > or =29 degrees C was considered to be heat stress (HS). Exposure to HS was defined as following: NH, no heat stress; HS1, exposure to at least 1 day of maximum temperature > or =29 degrees C and average daily maximum temperature (ADMT) <29 degrees C; and HS2, exposure to ADMT > or =29 degrees C. In Study 1, exposure of cows to HS1 and HS2 from 50 to 20 prior to AI was associated with reduced CR compared to cows not exposed to HS (28.8, 23.0, and 31.3%, respectively). Post-AI HS was not associated with CR. Cows inseminated following estrus detection or timed AI had similar CR. As the number of AI increased, CR decreased. Multiparous cows had lower CR than primiparous cows, and occurrence of milk fever and retained placenta was associated with decreased CR. In Study 2, PL was not associated with exposure to HS either prior to or after AI. Cows diagnosed with clinical mastitis experienced increased PL, but parity, number of AI, AI protocol, milk production, and days postpartum at AI were not associated with PL. In conclusion, CR was affected by HS prior to AI, parity, number of AI, and postparturient diseases, whereas PL was affected by clinical mastitis.     

Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding

The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes. In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers. Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture. Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities. Galactose was shown to be a more critical nutrient when cultures reached a high density. In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth. These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose. The inverse was not true and led, at high densities, to a decrease of cell viability. In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium. VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures. These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection. Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.     

Cryptococcus neoformans varieties as agents of cryptococcosis in Brazil

The study of the clinical isolates of Cryptococcus neoformans from 83 Brazilian patients with disseminated cryptococcosis showed that 75 were C. neoformans var. neoformans and 8 were var. gattii. Twenty-seven isolates were serotyped; all 19 var. neoformans were serotype A and all 8 var. gattii were serotype B. The correlation of the varieties of C. neoformans with the presence or not of hosts predisposing conditions to the mycosis showed that: (1) cryptococcosis caused by gattii variety occurred in 7 (58.3%) of the 12 nonimmunosuppressed patients, and (2) cryptococcosis caused by neoformans variety occurred in 65 (98.5%) of the 66 AIDS patients and in all 5 patients with other immunosuppressive conditions. The comparison of the distribution of the gattii and neoformans varieties between the nonimmunosup-pressed and immunosuppressed patients showed a significant statistical difference (p < 0.01).     

Pathogenicity of Epicoccum sorghinum towards dragon fruits (Hylocereus species) and in vitro evaluation of chemicals with antifungal activity

Recent reports of plant diseases that result in yield reduction and increasing demand for dragon fruits raise concerns of fruit supply shortage. Emerging plant diseases may play an important role in increasing yield losses and reducing the availability of stem cuttings (source of planting materials). Understanding the aetiology of current and new diseases of dragon fruit is important to address production issues and to formulate effective disease control measures. This study reports Epicoccum sorghinum as a potential emerging pathogen of dragon fruit. Epicoccum sorghinum MBDF0024a was isolated from dragon fruit stems (Hylocereus monacanthus) showing brown spot symptoms. DNA sequence of the internal transcribed spacer (ITS-rDNA), beta tubulin and actin gene regions of fungal isolate MBDF0024a had high similarities to E. sorghinum stains. Epicoccum sorghinum MBDF0024a was pathogenic to three cultivated dragon fruit species (Hylocereus undatus, H. monacanthus and H. megalanthus) in repeated laboratory and glasshouse trials. Large brown lesions developed on 3-week-old inoculated rooted stem cuttings 3 days postinoculation (dpi). Yellowing of the lesion (advance part) started at five dpi, and at seven dpi, yellowing was observed in the stem. As there are no reported control measures for diseases caused by E. sorghinum, this study screened chemicals with antifungal properties. A biopesticide containing B. subtilis (2 ml/400 ml), and chemicals isoprothiolane (2.25 ml/400 ml), mancozeb (2 g/400 ml) and pyraclostrobin (1 ml/400 ml) (chemical control) completely inhibited the in vitro growth of E. sorghinum MBDF0024a. The results establish E. sorghinum as a new and emerging pathogen of dragon fruit that could be a major yield-limiting disease if left uncontrolled. The biopesticide can be considered a fairly safe option for disease management, but glasshouse and field studies are needed for validation.     

Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings

The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between (15)N and (1)H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with phi and psi dihedral angles favoring the beta or P(II) regions. Each statistical segment has a highly anisotropic shape, with the N-H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the acid-denatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.     

Genome-wide Introgression Lines and their Use in Genetic and Molecular Dissection of Complex Phenotypes in Rice (Oryza sativa L.)

Tremendous efforts have been taken worldwide to develop genome-wide genetic stocks for rice functional genomic (FG) research since the rice genome was completely sequenced. To facilitate FG research of complex polygenic phenotypes in rice, we report the development of over 20 000 introgression lines (ILs) in three elite rice genetic backgrounds for a wide range of complex traits, including resistances/tolerances to many biotic and abiotic stresses, morpho-agronomic traits, physiological traits, etc., by selective introgression. ILs within each genetic background are phenotypically similar to their recurrent parent but each carries one or a few traits introgressed from a known donor. Together, these ILs contain a significant portion of loci affecting the selected complex phenotypes at which allelic diversity exists in the primary gene pool of rice. A forward genetics strategy was proposed and demonstrated with examples on how to use these ILs for large-scale FG research. Complementary to the genome-wide insertional mutants, these ILs opens a new way for highly efficient discovery, candidate gene identification and cloning of important QTLs for specific phenotypes based on convergent evidence from QTL position, expression profiling, functional and molecular diversity analyses of candidate genes, highlights the importance of genetic networks underlying complex phenotypes in rice that may ultimately lead to more complete understanding of the genetic and molecular bases of quantitative trait variation in rice. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s11103-005-8519-3