Phenotypic characterization and 16S rDNA identification of culturable non-obligate halophilic bacterial communities from a hypersaline lake, La Sal del Rey, in extreme South Texas (USA)

Background: La Sal del Rey ( the King’s Salt”) is one of several naturally-occurring salt lakes in Hidalgo County, Texas and is part of the Lower Rio Grande Valley National Wildlife Refuge. The research objective was to isolate and characterize halophilic microorganisms from La Sal del Rey. Water samples were collected from the lake and a small creek that feeds into the lake. Soil samples were collected from land adjacent to the water sample locations. Sample salinity was determined using a refractometer. Samples were diluted and cultured on a synthetic saline medium to grow halophilic bacteria. The density of halophiles was estimated by viable plate counts. A collection of isolates was selected, gram-stained, tested for catalase, and characterized using API 20E® test strips. Isolates were putatively identified by sequencing the 16S rDNA. Carbon source utilization by the microbial community from each sample site was examined using EcoPlate™ assays and the carbon utilization total activity of the community was determined. Results: Results showed that salinity ranged from 4 parts per thousand (ppt) at the lake water source to 420 ppt in water samples taken just along the lake shore. The density of halophilic bacteria in water samples ranged from 1.2 × 102 - 5.2 × 103 colony forming units per ml (cfu ml-1) whereas the density in soil samples ranged from 4.0 × 105 - 2.5 × 106 colony forming units per gram (cfu g-1). In general, as salinity increased the density of the bacterial community decreased. Microbial communities from water and soil samples were able to utilize 12 - 31 carbon substrates. The greatest number of substrates utilized was by water-borne communities compared to soil-based communities, especially at lower salinities. The majority of bacteria isolated were gram-negative, catalase-positive, rods. Biochemical profiles constructed from API 20E® test strips showed that bacterial isolates from low-salinity water samples (4 ppt) showed the greatest phenotypic diversity with regards to the types and number of positive tests from the strip. Isolates taken from water samples at the highest salinity (420 ppt) tended to be less diverse and have only a limited number of positive tests. Sequencing of 16S DNA displayed the presence of members of bacterial genera Bacillus, Halomonas, Pseudomonas, Exiguobacterium and others. The genus Bacillus was most commonly identified. None of the isolates were members of the Archaea probably due to dilution of salts in the samples. Conclusions: The La Sal del Rey ecosystem supports a robust and diverse bacterial community despite the high salinity of the lake and soil. However, salinity does appear to a limiting factor with

Astrocytes Play a Key Role in <i>Drosophila</i> Mushroom Body Axon Pruning

<div><p>Axon pruning is an evolutionarily conserved strategy used to remodel neuronal connections during development. The <i>Drosophila</i> mushroom body (MB) undergoes neuronal remodeling in a highly stereotypical and tightly regulated manner, however many open questions remain. Although it has been previously shown that glia instruct pruning by secreting a TGF-β ligand, myoglianin, which primes MB neurons for fragmentation and also later engulf the axonal debris once fragmentation has been completed, which glia subtypes participate in these processes as well as the molecular details are unknown. Here we show that, unexpectedly, astrocytes are the major glial subtype that is responsible for the clearance of MB axon debris following fragmentation, even though they represent only a minority of glia in the MB area during remodeling. Furthermore, we show that astrocytes both promote fragmentation of MB axons as well as clear axonal debris and that this process is mediated by ecdysone signaling in the astrocytes themselves. In addition, we found that blocking the expression of the cell engulfment receptor Draper in astrocytes only affects axonal debris clearance. Thereby we uncoupled the function of astrocytes in promoting axon fragmentation to that of clearing axonal debris after fragmentation has been completed. Our study finds a novel role for astrocytes in the MB and suggests two separate pathways in which they affect developmental axon pruning.</p></div

Knocking down the ecdysone regulated <i>drpr</i> or inhibiting endocytosis in astrocytes results in uncleared axonal debris.

<p>Confocal Z-projections of adult (A–D) or 6APF brains (E, F) expressing CD8-GFP (A, E), additionally expressing Drpr-RNAi (B, C), <i>Shi^ts1</i> (D), or EcR-DN (F) in astrocytes using the alrm-Gal4 driver. High and prolonged expression of Drpr-RNAi (C) was achieved by the Tub>GAL4 flipout system (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086178#pone.0086178.s003" target="_blank">Fig. S3A</a> for details) (A₂–D₂) Represent a higher-magnification of the dorsal tip. No ectopic γ axon branches were detected in adults when astrocytic engulfment ability was impaired with expression of <i>Drpr</i> RNAi (B₁, C₁) or <i>shibire^ts</i> (D₁). Uncleared debris (arrowhead) was detected in 100% of flies of the tested transgenes (<i>Drpr</i> RNAi, 12 flies, B₂; forced expressed <i>Drpr</i> RNAi, 12 flies, C₂ and <i>shibire^ts</i>, 17 flies, D₂). (E, F) Drpr staining in WT (E) and flies expressing EcR-DN in astrocytes (F) at 6APF. Marked box represents regions that were used fort the quantification in G. (G) EcR-DN expressing flies show significantly lower staining for Drpr (0.16±0.01, n = 14) than WT flies (12.88±1.37, n = 10, p<0.001) at 6APF (quantification was done on confocal single slices). Grey represent anti-FasII staining in A–D and in E₁–F₁ and Drpr staining in E₂–F₂. Green in E and F represents alrm-GAL4 driven mCD8::GFP. Red in E_3–4 and F_3–4 is Drpr staining. The scale bars are 20 μm. Genotypes: (A) y,w;alrm-GAL4/+;alrm-GAL4,mCD8::GFP/+; (B) y,w;alrm-GAL4/+;alrm-GAL4,mCD8::GFP,Dcr2/UAS-Drpr-RNAi; (C) y,w;P{GAL4-αTub84B(FRT.CD2).P}/UAS-FLP;alrm-GAL4,mCD8::GFP,Dcr2/UAS-Drpr-RNA (D) y,w;alrm-GAL4/+;alrm-GAL4,mCD8::GFP/UAS-Shit^ts. (E) y,w; alrm-GAL4/+;alrm-GAL4,mCD8::GFP/+ (F) y,w; alrm-GAL4/UAS-EcR-DN;alrm-GAL4,mCD8::GFP/+.</p

Astrocyte's <i>Ecdysone Receptor</i> controls multiply aspects of MB γ neuron remodeling.

<p>Confocal Z-projections of brains expressing CD8-GFP driven by alrm-Gal4 (A, C, E, G, I), or those additionally expressing EcR-DN (B, D, F, H, J) or myo in addition to EcR-DN (K) in astrocytes at 6 h APF (A, B), 18 h APF (C, D), 24 h APF (E, F) and Adult (G–K) reared at either 25°C (A–H) or reared at 18°C until late larval stage and then transferred to 29°C until eclosion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086178#pone.0086178.s002" target="_blank">Fig. S2B</a>, I–K). (A,B) At 6 h APF astrocytes expressing EcR-DN do not infiltrate the MB γ lobes (see insets). Overexpression of EcR in astrocytes results in a delay in axon pruning at 18 h APF (D compare to C) and at 24 h APF (F compare to E) that persisted into adult (H compare to G). Elevated expression in astrocytes (I–J; see S2A for details) resulted in more persistent unpruned axons. Overexpression of Myo in addition to EcR-DN (K) partially rescued both the fragmentation (L) and the debris clearance (M) defects. Quantification was performed by ranking analyses that are detailed in the materials and methods section. * P<0.05; ** P<0.005. Bars represent SEM. Grey in A₁–K₁ and magenta in A₂–K₂ represents anti-FasII staining. Green in A₂–K₂ represents alrm-GAL4 driven mCD8::GFP. Scale bar is 20 μM. Genotypes: (A–I): y,w; alrm-GAL4/+;alrm-GAL4,mCD8::GFP/+ (B–J): y,w; alrm-GAL4/UAS-EcR-DN;alrm-GAL4,mCD8::GFP/+. (K): y,w; alrm-GAL4/UAS-EcR-DN;alrm-GAL4,mCD8::GFP/UAS-Myo.</p

Astrocytes infiltrate and engulf degenerating MB γ neurons at 6h APF.

<p>(A–C) Confocal Z-projections of brains expressing alrm-GAL4 driven mCD8::GFP focusing on the MB medial (A) or dorsal (B) lobes or the peduncle (C) at 6 h APF as depicted by the cartoons on the left (A₁–C₁). Fas II staining shows clear spherical holes devoid of FasII expression in the MB γ dorsal (A₂) and medial (B₂) lobes and a slight decrease in FasII staining in the peduncle (C₃). These holes are occupied by astrocyte extensions (A₃–C₃) infiltrating into the MB axon bundle (arrows in A₄–C₄). Magenta represents anti-FasII staining. Green is alrm-GAL4 driven mCD8::GFP. (D-E) Confocal single slices of the MB dorsal lobe tip (D) and the peduncle (E). While γ neurons were labeled by the Q-system (ET40-QF driving QUAS-mtdT-3xHA expression), astrocytes were labeled by alrm-GAL4 driven mCD8::GFP expression. At 6 h APF, γ axons are largely fragmented at the dorsal tip lobe (D₂) but only few fragments are detected at the peduncle (E₂). While astrocytes infiltrate both the dorsal lobe (D₄) and the peduncle (E₄), clear engulfment events can only be seen around the dorsal tip (D₄). Red is QF-ET40 driven QUAS-mtdt-3xHA. Green is alrm-GAL4 driven mCD8::GFP. The scale bars are 20 μm. Genotypes: (A–C) w;alrm-GAL4;alrm-GAL4,UAS-mCD8::GFP/+ (D–E) y,w;QF-ET40,QUAS-mtdT-3xHA/+;alrm-GAL4,UAS-mCD8::GFP/+.</p

Astrocytes are necessary for efficient axon pruning.

<p>(A–B) Confocal Z-projections of adult brains expressing CD8-GFP (A) or additionally UAS-DTI (diphtheria toxin; B) driven by alrm-Gal4. Driving the expression of DTI in astrocytes resulted in their partial ablation (grey, A_2, B₂) and results in fragmentation defects (arrow in B₁) and uncleared debris (arrowhead in B₁) in escapers (13 out of 118 expected flies). Grey in A₁–B₁ and magenta in A₃–B₃ represents anti-FasII. Grey in A₂–B₂ and green in A₃–B₃ are alrm-GAL4 driven mCD8::GFP. The scale bar is 20 μm. Genotypes: (A) y,w;Sp/CyO;alrm-GAL4,UAS-mCD8::GFP/+; (B) y,w;UAS-DTI/+;alrm GAL4, UAS-mCD8::GFP/+.</p

Astrocytes surround the MB during developmental axon pruning.

<p>(A) Scheme of developmental pruning of MB γ neurons. During larval development and up to puparium formation (0 h APF), γ neurons extend a single process that sends out dendrites near the cell body and continues as an axon peduncle that bifurcates to form a dorsal and a medial branch. At the onset of puparium, a glial derived TGF-β signal induces the expression of the ecdysone receptor-B1 (EcR-B1) within γ neurons. Subsequently, an ecdysone pulse activates EcR-B1, resulting in a largely unknown transcriptional cascade. At 6 h APF both axonal branches, as well as the dendrites begin to undergo fragmentation, while the pedunclar axon remains intact and the neurons retain their cell body. The fragmented axons are then cleared by glia that surround these axons. Subsequently, γ neurons regrow axons that project into a new, adult specific and medially projecting lobe. (B–E) Confocal Z-projections of brains expressing alrm-GAL4 driving mCD8::GFP at 0 h APF (B), 6 h APF (C), 18 h APF (D) or 24 h APF (E). (B) At the onset of metamorphosis (0 h APF) astrocytic membranes are evenly dispersed in the region of the MB lobes (higher magnification in A₂). (C) By 6 h APF, at the onset of pruning, the astrocytes have changed their morphology and have begun to infiltrate the degenerating lobes. (D) At 18 h astrocyte membranes surround axon fragments (arrowhead in D₂). By 18 h to (E) 24 h APF there is a significant decrease in alrm-GAL4 positive membranes. Newly extended axon branches of α/β neurons, are also stained with anti-FasII antibody at 24 h APF (E). Magenta represents anti-FasII staining. Green is alrm-GAL4 driven mCD8::GFP. The scale bars are 20 μm. Genotypes: (B–E) y,w;alrm-GAL4;alrm-GAL4,UAS-mCD8::GFP/+.</p

The number of astrocytes at the MB remains constant throughout remodeling.

<p>(A–E) Confocal Z-projections of alrm-GAL4 driven RedStinger expression (red, A–E) labeling astrocytes nuclei at 0 h APF (A), 6 h APF (B, E), 18 h APF (C) or 24 h APF (D). (A–D) Labeling astrocyte cell bodies shows that the number of the astrocytes does not significantly change during remodeling. (E) High magnification view of dorsal tip reveals that astrocytes (red) label only part of the repo⁺ nuclei in the vicinity of the MB lobe tip. (F) To characterize this quantitatively, we counted the number of astrocytes nuclei within 5 μM of the dorsal tip and found that only 1–2 astrocytes are located near the dorsal tip throughout remodeling. Magenta represents anti-FasII and anti-Repo staining. Red is alrm-GAL4 driven RedStinger. (G) Confocal Z-projections (G) or single slices (individual cells) of brains expressing alrm-Gal4 driven CD8-GFP in just a few cells (see materials and methods). While the 6 glial cells proximal to the dorsal tip are not labeled with mCD8::GFP (close-up of dorsal tip, region 5), four cells that are located further away from the lobe are (see close-ups of cell 1–4). Grey represents FasII and Repo staining (G₁ and upper panels in close-ups), DAPI staining in the lower panels in close ups or alrm-Gal4 driven CD8-GFP (G₂ and middle panels in close-ups). Magenta represents FasII and Repo staining and green represents alrm-Gal4 driven CD8-GFP in H₃ and lower panels in close-ups. The scales bars are 20 μm. Genotypes: (A–E): w;UAS-RedStinger/+;alrm-GAL4/+. (G) y,w,hsFlp,FRT19A/y,w,hsFlp,19A,tubP-GAL80;alrm-GAL4;alrm-GAL4,UAS-mCD8::GFP.</p

Developmental Coordination during Olfactory Circuit Remodeling in Drosophila

Developmental neuronal remodeling is crucial for proper wiring of the adult nervous system. While remodeling of individual neuronal populations has been studied, how neuronal circuits remodel-and whether remodeling of synaptic partners is coordinated-is unknown. We found that the Drosophila anterior paired lateral (APL) neuron undergoes stereotypic remodeling during metamorphosis in a similar time frame as the mushroom body (MB) gamma-neurons, with whom it forms a functional circuit. By simultaneously manipulating both neuronal populations, we found that cell-autonomous inhibition of gamma-neuron pruning resulted in the inhibition of APL pruning in a process that is mediated, at least in part, by Ca2+-Calmodulin and neuronal activity dependent interaction. Finally, ectopic unpruned MB gamma axons display ectopic connections with the APL, as well as with other neurons, at the adult, suggesting that inhibiting remodeling of one neuronal type can affect the functional wiring of the entire micro-circuit