Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-beta and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought.     

Silicon chip-based patch-clamp electrodes integrated with PDMS microfluidics

We report on a silicon wafer-based device that can be used for recording macroscopic ion channel protein activities across a diverse group of cell-types. Gigaohm seals were achieved for CHO-K1 and RIN m5F cells, and both cell-attached and whole-cell mode configurations were also demonstrated. Two distinct intrinsic potassium ion channels were recorded in whole-cell mode for HIT-T15 and RAW 264.7 cells. Polydimethylsiloxane (PDMS) microfluidics were also coupled with the micromachined silicon chips in order to demonstrate that a single cell could be selectively directed to a micropore, and membrane protein currents could subsequently be recorded. These silicon chip-based devices have significant advantages over traditional micropipette approaches, and may serve as combinatorial tools for investigating membrane biophysics, pharmaceutical screening, and other bio-sensing tasks.     

Population dynamics and effects of Oebalus ornatus (Hemiptera: Pentatomidae) on rice yield and quality in southwestern Colombia

Field experiments were conducted from 1989 to 1992 to determine the effects of Oebalus ornatus (Sailer) on Cica 8 rice, Oryza sativa L., yield and to study the population dynamics of the insect. The effect of O. ornatus was measured for seven population levels (0, 2, 3, 4, 8, 12, and 24 sexed pairs per 20 panicles) at three stages of grain development (flowering, milk, and soft dough stage). Insect feeding during the flowering and milk stages of grain development caused more damage than feeding during the soft dough stage. The action threshold was calculated to be 14 O. ornatus adults per square meter for the flowering and milk stages and 67 for the soft dough stage of grain maturity. Population densities that would reduce rice yield in southwestern Colombia were not observed during the 3 yr of the study.     

A non-destructive method based on the polymerase chain reaction for detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp

Current methods to detect hepatopancreatic parvovirus (HPV) infection of penaeid shrimp depend on invasive techniques that require dissecting the organs infected by this virus. However, sacrificing valuable stocks in order to determine their HPV status can be a drawback in the case of breeding programs. A method was developed for HPV detection by applying a polymerase chain reaction (PCR) assay to fecal samples collected from live HPV-infected shrimp Penaeus chinensis. A pair of PCR primers, 1120F/1120R, which amplify a 592 base pair (bp) region from the virus genome, was designed from previously known HPV sequence information (HPV clone HPV8). PCR amplification with these primers generated a product of the expected size directly from the crude feces of HPV-infected shrimp but not from the feces of specific pathogen-free (SPF) shrimp. The HPV origin of the amplified product was validated by means of an in situ hybridization assay where the product of the amplification, labeled with digoxigenin (DIG)-11-dUTP, showed an intense reaction within hepatopancreatic cells displaying characteristic HPV lesions on HPV-infected shrimp. No reaction to this probe was observed when reacted in situ with sections of the hepatopancreas of SPF specimens or to sections of shrimp infected by the infectious hypodermal and hematopoietic necrosis virus (IHHNV), another parvovirus of penaeid shrimp. These primers were tested for specificity against homologous and nonhomologous viruses and no product was amplified. A fragment of the expected size was obtained only when purified HPV or purified HPV8 plasmid was used as template DNA. Under optimized conditions, these primers detected as little as 1 fg of purified HPV8 plasmid DNA, equivalent to approximately 300 HPV particles. Analysis of fecal samples by PCR may prove useful for non-lethal screening of valuable shrimp of unknown HPV status. This same strategy also might be used for detection of other enteric viruses that infect penaeid shrimp.     

Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection

An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue-green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e-124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.     

Parasites component community in wild population of Pterophyllum scalare Schultze, 1823 and Mesonauta acora Castelnau, 1855, cichlids from the Brazilian Amazon

The aim of the present study was to compare the component parasite communities of the Pterophyllum scalare and Mesonauta acora cichlids in the Amazon River system in northern Brazil. From September to December 2012, 42 specimens of P. scalare and 38 specimens of M. acora were captured using hand nets and gillnets in the Igarapé Fortaleza basin, a tributary of the Amazon River in the state of Amapá. Of the P. scalare specimens examined, 97.6% were parasitized by Ichthyophthirius multifiliis, Tripartiella sp., Trichodina nobilis, Gussevia spiralocirra, Posthodiplostomum sp., Capillaria pterophylli, Ichthyouris sp. and Gorytocephalus spectabilis. Similarly, all specimens of M. acora were parasitized by I. multifiliis, Tripartiella sp, T. nobilis, Sciadicleithrum joanae, Posthodiplostomum sp., Pseudoproleptus sp., Ichthyouris sp. and G. spectabilis. However, for both hosts the dominance was of I. multifiliis and with an overdispersion of parasites. Parasite communities of P. scalare and M. acora were similar and only Pseudoproleptus sp. and Posthodiplostomum sp. were larvae. Brillouin diversity, parasite species richness and evenness were higher for M. acora than for P. scalare, which presented a negative correlation of parasite abundance with body size. Both cichlid species had parasite communities characterized by low diversity and low species richness, with a predominance of ectoparasite species and greatest richness of helminth species, with a low abundance of endoparasites. This was the first study on the parasite diversity in wild P. scalare and M. acora.     

Juxtaposition and Disturbance: Disentangling the Determinants of Lizard Community Structure

Disturbance alters the structure and dynamics of communities. Here, we examined the effects of seasonal flooding on the lizard community structure by comparing two adjacent habitats, a seasonally flooded and a non‐flooded forest, in a Cerrado–Amazon ecotone area, the Cantão State Park, Tocantins state, Brazil. Despite the strong potential impact of seasonal flooding, the only significant environmental difference detected was more termite mounds in non‐flooded forests. Species richness was significantly higher in the non‐flooded forest. Colobosaura modesta, followed by Mabuya frenata and Anolis brasiliensis, were the only species that differed in number of captures between sites. Colobosaura modesta was exclusively found in the non‐flooded forest, while Anolis brasiliensis was the most captured in the flooded forest. Mabuya frenata is indicated as an indicator species in the flooded forest, and Colobosaura modesta in the non‐flooded forest. We found a significant association between lizard abundances and habitat characteristics, with flooding, canopy cover, and logs being the best predictors. A phylogenetic community structure analysis indicated a lack of structure in both lizard assemblages. Overall, we show that seasonal flooding can strongly impact species richness and species occurrence patterns, but not phylogenetic community structure. The Amazon–Cerrado transition is undergoing pronounced transformations due to deforestation and climate change. Despite being species‐poor compared with central areas in Amazon or Cerrado, this ecotone harbors species with important adaptations that could hold the key to persistence in human‐disturbed landscapes or during periods of climate change.     

Non-Invasive Measurement of Adrenocortical Activity in Blue-Fronted Parrots (Amazona aestiva,Linnaeus, 1758)

Parrots kept in zoos and private households often develop psychological and behavioural disorders. Despite knowing that such disorders have a multifactorial aetiology and that chronic stress is involved, little is known about their development mainly due to a poor understanding of the parrots’ physiology and the lack of validated methods to measure stress in these species. In birds, blood corticosterone concentrations provide information about adrenocortical activity. However, blood sampling techniques are difficult, highly invasive and inappropriate to investigate stressful situations and welfare conditions. Thus, a non-invasive method to measure steroid hormones is critically needed. Aiming to perform a physiological validation of a cortisone enzyme immunoassay (EIA) to measure glucocorticoid metabolites (GCM) in droppings of 24 Blue-fronted parrots (Amazona aestiva), two experiments were designed. During the experiments all droppings were collected at 3-h intervals. Initially, birds were sampled for 24 h (experiment 1) and one week later assigned to four different treatments (experiment 2): Control (undisturbed), Saline (0.2 mL of 0.9% NaCl IM), Dexamethasone (1 mg/kg IM) and Adrenocorticotropic hormone (ACTH; 25 IU IM). Treatments (always one week apart) were applied to all animals in a cross-over study design. A daily rhythm pattern in GCM excretion was detected but there were no sex differences (first experiment). Saline and dexamethasone treatments had no effect on GCM (not different from control concentrations). Following ACTH injection, GCM concentration increased about 13.1-fold (median) at the peak (after 3–9 h), and then dropped to pre-treatment concentrations. By a successful physiological validation, we demonstrated the suitability of the cortisone EIA to non-invasively monitor increased adrenocortical activity, and thus, stress in the Blue-fronted parrot. This method opens up new perspectives for investigating the connection between behavioural disorders and stress in this bird species, and could also help in their captive management.     

Phosphorus remobilization from rice flag leaves during grain filling: an RNA‐seq study

The physiology and molecular regulation of phosphorus (P) remobilization from vegetative tissues to grains during grain filling is poorly understood, despite the pivotal role it plays in the global P cycle. To test the hypothesis that a subset of genes involved in the P starvation response are involved in remobilization of P from flag leaves to developing grains, we conducted an RNA‐seq analysis of rice flag leaves during the preremobilization phase (6 DAA) and when the leaves were acting as a P source (15 DAA). Several genes that respond to phosphate starvation, including three purple acid phosphatases (OsPAP3, OsPAP9b and OsPAP10a), were significantly up‐regulated at 15 DAA, consistent with a role in remobilization of P from flag leaves during grain filling. A number of genes that have not been implicated in the phosphate starvation response, OsPAP26, SPX‐MFS1 (a putative P transporter) and SPX‐MFS2, also showed expression profiles consistent with involvement in P remobilization from senescing flag leaves. Metabolic pathway analysis using the KEGG system suggested plastid membrane lipid synthesis is a critical process during the P remobilization phase. In particular, the up‐regulation of OsPLDz2 and OsSQD2 at 15 DAA suggested phospholipids were being degraded and replaced by other lipids to enable continued cellular function while liberating P for export to developing grains. Three genes associated with RNA degradation that have not previously been implicated in the P starvation response also showed expression profiles consistent with a role in P mobilization from senescing flag leaves.