Spaceflight Causes Increased Virulence of Serratia Marcescens on a Drosophila Melanogaster Host

Drosophila melanogaster, or the fruit fly, has long been an important organism for Earth-based research, and is now increasingly utilized as a model system to understand the biological effects of spaceflight. Studies in Drosophila melanogaster have shown altered immune responses in 3rd instar larvae and adult males following spaceflight, changes similar to those observed in astronauts. In addition, spaceflight has also been shown to affect bacterial physiology, as evidenced by studies describing altered virulence of Salmonella typhimurium following spaceflight and variation in biofilm growth patterns for the opportunistic pathogen Pseudomonas aeruginosa during flight. We recently sent Serratia marcescens Db11, a Drosophila pathogen and an opportunistic human pathogen, to the ISS on SpaceX-5 (Fruit Fly Lab-01). S. marcescens samples were stored at 4degC for 24 days on-orbit and then allowed to grow for 120 hours at ambient station temperature before being returned to Earth. Upon return, bacteria were isolated and preserved in 50% glycerol or RNAlater. Storage, growth, and isolation for ground control samples were performed using the same procedures. Spaceflight and ground samples stored in 50% glycerol were diluted and injected into 5-7-day-old ground-born adult D. melanogaster. Lethality was significantly greater in flies injected with the spaceflight samples compared to those injected with ground bacterial samples. These results indicate a shift in the virulence profile of the spaceflight S. marcescens Db11 and will be further assessed with molecular biological analyses. Our findings strengthen the conclusion that spaceflight impacts the virulence of bacterial pathogens on model host organisms such as the fruit fly. This research was supported by NASA's ISS Program Office (ISSPO) and Space Life and Physical Sciences Research and Applications (SLPSRA)

<i>Rhomboid</i> Enhancer Activity Defines a Subset of <i>Drosophila</i> Neural Precursors Required for Proper Feeding, Growth and Viability

<div><p>Organismal growth regulation requires the interaction of multiple metabolic, hormonal and neuronal pathways. While the molecular basis for many of these are well characterized, less is known about the developmental origins of growth regulatory structures and the mechanisms governing control of feeding and satiety. For these reasons, new tools and approaches are needed to link the specification and maturation of discrete cell populations with their subsequent regulatory roles. In this study, we characterize a <i>rhomboid</i> enhancer element that selectively labels four <i>Drosophila</i> embryonic neural precursors. These precursors give rise to the hypopharyngeal sensory organ of the peripheral nervous system and a subset of neurons in the deutocerebral region of the embryonic central nervous system. Post embryogenesis, the <i>rhomboid</i> enhancer is active in a subset of cells within the larval pharyngeal epithelium. Enhancer-targeted toxin expression alters the morphology of the sense organ and results in impaired larval growth, developmental delay, defective anterior spiracle eversion and lethality. Limiting the duration of toxin expression reveals differences in the critical periods for these effects. Embryonic expression causes developmental defects and partially penetrant pre-pupal lethality. Survivors of embryonic expression, however, ultimately become viable adults. In contrast, post-embryonic toxin expression results in fully penetrant lethality. To better define the larval growth defect, we used a variety of assays to demonstrate that toxin-targeted larvae are capable of locating, ingesting and clearing food and they exhibit normal food search behaviors. Strikingly, however, following food exposure these larvae show a rapid decrease in consumption suggesting a satiety-like phenomenon that correlates with the period of impaired larval growth. Together, these data suggest a critical role for these enhancer-defined lineages in regulating feeding, growth and viability.</p></div

Temporal control of toxin expression separates defects in development and viability.

<p>(A-B) Temperature-sensitive Gal80 expression controls Gal4-dependent GFP reporter activity. Stage 17 embryos, <i>z</i>-projected dorsal views. (C-D) Quantification of pupae formed (C) and surviving to adulthood (D) for larvae subject to different toxin expression conditions. (E-G) Analysis of age at pupariation (E), length of pupation (F) and age at eclosion (G) for non-toxin-targeted and embryonically-targeted flies. Since developmental timing is temperature dependent, data are shown as experimentals (<i>UAS-DTI</i> positive) normalized to controls (<i>UAS-DTI</i> negative). *p<0.01 or **p<0.001. (H) Comparison of non-targeted and embryonically-targeted adults. Scale bar, 1 mm. (I) Quantification of wing area for non-targeted and embryonically-targeted flies. <i>p</i>>0.1 for inter-sex comparisons.</p

<i>rho</i> enhancer activity maps to four neural precursors in the embryonic head.

<p>(A) The <i>rho</i> locus highlighting the Rho654 enhancer’s four conserved elements (RhoA-RhoD). (B-D) Immunostaining of <i>Rho654>H2B-YFP</i> (B), <i>RhoBB>H2B-YFP</i> (C) and <i>RhoAAA>H2B-YFP</i> (D) reporter lines demonstrating that the RhoB region of Rho654 mediates activity in four neural precursors within the head (arrowheads in B and C) while the RhoA region acts in abdominal SOPs (A1 denotes first the first abdominal segment in B and D). Early (C) and slightly later (B,D) stage 11, <i>z</i>-projected lateral views. (E-M) Comparison of <i>Rho654>H2B-YFP</i> (E-L) with published expression data for CNS neuroblasts and SOPs (M, adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134915#pone.0134915.ref017" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134915#pone.0134915.ref090" target="_blank">90</a>]). Co-expression of YFP with Dpn (E) indicates neural precursor identity. Lack of YFP co-expression with Msh (F), Ind (G), Vnd (H), Otd (I), or Fas2 (J) restricts Rho654 cell (green borders) identities to the Dv1/3 SOPs and Dv4/7 neuroblasts. Co-expression of YFP with DPax2 (K) and Ato (L) (arrowheads) is consistent with expression data for the Dv1/3 SOPs (M). Stage 11, <i>z</i>-projected ventral views with anterior up. PC: protocerebrum, DC: deutocerebrum, TC: tritocerebrum, VNC: ventral nerve cord. Bold dashed lines represent approximate neuromere boundaries.</p

Fate mapping and characterization of labeled neural precursor progeny.

<p>(A-C) Lateral (A) and ventral (B-C) views of <i>Rho654>H2B-YFP</i>-labeled Dv1/3/4/7 progeny migrating posteromedially along the ventral pharynx at stage 12. Subsets of these cells express DPax2 and all express pERK (C, green outline denotes position of <i>Rho654>H2B-YFP</i>-labeled cells). <i>z</i>-projections with anterior to the left. (D-H) At stage 17, YFP-labeled cells form the HPSO (D-E, arrowhead) and a bilaterally symmetric subset of deutocerebral CNS neurons (D-E, arrow). Subsets of HPSO cells (solid outlines) express an <i>ato</i> reporter (F), DPax2 (G) and Eys (H). None of these markers is expressed in the YFP-labeled CNS-neurons (dashed outlines). <i>z</i>-projected dorsal views. (I-L) Expression of Eys remains in heterozygous <i>rho</i>^<i>7M43</i> or <i>Spi</i>^<i>1</i> mutants (I,K, arrowheads) but is lost in homozygotes (J,L, open arrowheads). Stage 17, <i>z</i>-projected dorsal views. (M-P) Rho654 reporter activity is not detectable in the pharyngeal epithelial region of first instar larvae (M-N) but is detectable by third instar (O-Q). The PPS (successor of the HPSO) is labeled by the neuronal markers Futsch (marked by monoclonal antibody 22C10) and Elav. <i>z</i>-projections lateral views with anterior to the left and dorsal up. (R) Rho654 reporter activity is also detected in a subset of midline cells in the supraesophageal CNS as well as a network of projections within the ventral nerve cord of third instar larvae. Bruchpilot (Brp, marked by nc82 antibody) defines the boundaries of the CNS neuropile. <i>z</i>-projected dorsal view. (S-T) Targeted overexpression of Aos alters PPS morphology, increasing the distance between Futsch-positive termini (asterisks, T) and Elav-positive nuclei (compare with Fig 2P-2Q). (U-V) Targeted overexpression of Rho produces dramatic effects on PPS morphology based on both abnormal Futsch-positive neuronal processes (V, asterisks) and the increase in Elav-positive nuclei. <i>z</i>-projected lateral views with anterior to the left and dorsal up.</p

Enhancer-targeted toxin expression impairs development and viability.

<p>(A) Eys expression is not detected in the HPSO of toxin-targeted embryos despite the persistence of reporter activity in Rho654-defined cells. Open arrowheads denote normal location of Eys-expressing cells (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134915#pone.0134915.g002" target="_blank">Fig 2H</a>). Stage 17, <i>z</i>-projected dorsal view. (B-E) Reporter expression is undetectable (B) or reduced (D) in the PPS/pharyngeal region of toxin-targeted larvae and the relative position of Futsch-positive PPS neuronal termini (asterisks, C and E) is altered (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134915#pone.0134915.g002" target="_blank">Fig 2M–2Q</a>). <i>z</i>-projected lateral views. (F) Comparison of control and toxin-targeted larvae at 72 hours AEL. Scale bar, 1 mm. (G) Quantification of length over larval/pupal time. (H) Age at pupariation for control and targeted flies. (I) Comparison of anterior spiracles in control and targeted pupae. (J) Quantification of abnormal anterior spiracle eversion. “Abnormal” denotes incomplete or absent eversion of at least one spiracle. (K) Pupal survival to adulthood. *p<0.01 or **p<0.001.</p