A Molecular Ecological Survey of Tick (Ixodes scapularis) Population Density and Their Infection Rates with Borrelia burgdorferi at the New Jersey School of Conservation

Studying vector populations of the Ixodes scapularis tick is of major importance in New Jersey, an endemic area of Lyme disease. This species of tick is a vector for various disease pathogens including the bacterium that causes Lyme disease, Borrelia burgdorferi. This study focused on Ixodes scapularis tick surveillance at the New Jersey School of Conservation, in Sussex County, New Jersey. Collection of host-seeking ticks began in July 2009 and has continued until December of 2010, by drag-cloth sampling in order to monitor tick abundances of all life-cycle stages. In 2010 relative abundances for larvae and nymphal stages declined as compared to the year 2009 data, as opposed to the adult stage where the relative abundances increased. In 2009, larvae Ixodes scapularis had a peak relative abundance in August of -0.145 ticks/m2 and in August of 2010 this decreased by 61% to -0.056 ticks/m2. Nymphal relative abundances decreased by 42%, in July 2009 the peak relative abundance was -0.036 ticks/m2 and in June 2010 peaked at -0.021 ticks/m2. In the fall, adult Ixodes scapularis ’ relative abundance peaked in November 2009 at -0.007 ticks/m2 and in October 2010 at -0.034 ticks/m2, showing a 486% increase. In the spring of 2010 adults from the 2009 fall that overwintered had an additional peak relative abundance at -0.006 ticks/m2. In addition, preliminary analyses of nymphal Ixodes scapularis infection with the bacterium Borrelia burgdorferi were conducted. An infection rate of -54.5% was found from a sample size of 11 nymphs. Although the infection rate of B. burgdorferi for the nymphal stage is the only available data for infection thus far, in the future, work will be conducted analyze infection for additional nymphs and adults as well as tests for other pathogens acquired and transmitted by this tick vector. This information as well as future analysis of the population trends and abundances during activity periods will benefit our understanding of the public health risks that the Ixodes scapularis tick poses in New Jersey and in other endemic areas for tick-borne diseases

Anoxia-Reoxygenation Regulates Mitochondrial Dynamics through the Hypoxia Response Pathway, SKN-1/Nrf, and Stomatin-Like Protein STL-1/SLP-2

<div><p>Many aerobic organisms encounter oxygen-deprived environments and thus must have adaptive mechanisms to survive such stress. It is important to understand how mitochondria respond to oxygen deprivation given the critical role they play in using oxygen to generate cellular energy. Here we examine mitochondrial stress response in <i>C. elegans</i>, which adapt to extreme oxygen deprivation (anoxia, less than 0.1% oxygen) by entering into a reversible suspended animation state of locomotory arrest. We show that neuronal mitochondria undergo DRP-1-dependent fission in response to anoxia and undergo refusion upon reoxygenation. The hypoxia response pathway, including EGL-9 and HIF-1, is not required for anoxia-induced fission, but does regulate mitochondrial reconstitution during reoxygenation. Mutants for <i>egl-9</i> exhibit a rapid refusion of mitochondria and a rapid behavioral recovery from suspended animation during reoxygenation; both phenotypes require HIF-1. Mitochondria are significantly larger in <i>egl-9</i> mutants after reoxygenation, a phenotype similar to stress-induced mitochondria hyperfusion (SIMH). Anoxia results in mitochondrial oxidative stress, and the oxidative response factor SKN-1/Nrf is required for both rapid mitochondrial refusion and rapid behavioral recovery during reoxygenation. In response to anoxia, SKN-1 promotes the expression of the mitochondrial resident protein Stomatin-like 1 (STL-1), which helps facilitate mitochondrial dynamics following anoxia. Our results suggest the existence of a conserved anoxic stress response involving changes in mitochondrial fission and fusion.</p></div

Anoxia induced mitochondrial oxidative stress.

<p>The fluorescence of MitoROGFP emitted at a 510(A,B) 405 nm or (C,D) 476 nm light from animals either under (A,C,E) normoxic conditions or (B,D,F) anoxic conditions. (E,F) Ratiometric images were generated from epifluorescence excited by 405 nm light relative to epifluorescence excited by 476 nm light. The ratio has been false colored with the indicated heat map, with high intensity indicative of ROGFP fluorescence in a more oxidative environment. (G) Quantification of mean light ratios evoked by the two excitation wavelengths at individual mitochondria from animals exposed to the given conditions. ANOVA followed by Dunnett's multiple comparison to animals exposed to normoxia (***p<0.001). N = 10–15 animals per condition. Error bars indicate SEM. Bar, 5 µm.</p

SKN-1 is required for anoxia-induced mitochondrial hyperfusion.

<p>The fluorescence of MitoGFP was observed along ventral cord neurites of (A,B,C) wild-type animals, (D,E,F) <i>skn-1(tm3411)</i> mutants, and (G,H,I) <i>egl-9(sa307) skn-1(tm3411)</i> double mutants under conditions of (A,D,G) normoxia, (B,E,H) following 24 hours of anoxia, or (C,F,I) following 3 hours of reoxygenation post-anoxia. (J,K) Quantification of the mean (J) length and (K) number of mitochondria along the ventral cord for the indicated genotypes and conditions. (L) Quantification of behavioral recovery (number of animals moving after 10 minutes of reoxygenation) of animals following 24 hours anoxia. Red bars indicate normoxia, blue bars indicate anoxia, and purple stippled bars indicate reoxygenation. ANOVA followed by Dunnett's multiple comparison to wild type, normoxia (***p<0.001, **p<0.01, *p<0.05). N = 15–35 animals per condition and/or genotype. Error bars indicate SEM. Bar, 5 µm.</p

Anoxia promotes suspended animation and eventually death in <i>C. elegans</i>.

<p>(A) Protocol for <i>C. elegans</i> anoxia treatment. The x-axis indicates time (in days since fertilization) and developmental stage (“EM” for embryo, “L2” and “L4” for respective larval stages, and “A1–A6” for the indicated day of mature adulthood). Boxes indicate the treatment during that particular period, with red indicating exposure to a normoxic environment (or the 1-day reoxygenation, labeled as “Re”) and blue indicating exposure to an anoxic environment. (B) Mean percentage of animals surviving after the given exposure time to anoxia. Error bars indicate SEM. (C) Mean percentage of animals moving (i.e., recovered from suspended animation) at the given time point following reoxygenation (post-anoxia). Individually plotted lines represent recovery following 12 (filled squares), 24 (filled triangles), 36 (empty triangles), and 48 (empty circles) hours of anoxia exposure. N = 15–35 animals per condition and/or genotype.</p

Model for anoxia-induced mitochondrial hyperfusion.

<p>Under conditions of normoxia in wild-type neurons, mitochondria undergo a balance of fission and fusion. Exposure to anoxia shifts the balance towards smaller and fewer mitochondria by promoting the canonical fission process. Reoxygenation shifts the balance back towards elongated mitochondria by promoting the canonical fusion process. Depending on the dual activities of the hypoxia response pathway (EGL-9 and HIF-1) and the oxidative stress pathway (SKN-1 and STL-1), reoxygenation can trigger hyperfusion, rapidly resulting in enlarged mitochondria. Mitochondrial dynamics in turn affect the suspended animation behavior of the animal. Hyperfused mitochondria, perhaps through a more efficient generation of ATP, allow neurons to rapidly resume function and rapidly re-emerge from suspended animation. Green ellipses indicate mitochondria distributed along neurites. Arrows indicate stimulatory interactions, whereas T-bars indicate inhibitory interactions.</p

Anoxia-induced mitochondrial hyperfusion requires the canonical mitochondrial fusion machinery.

<p>The fluorescence of MitoGFP was observed along ventral cord neurites of (A,B,C) wild-type animals, (D,E,F) <i>egl-9(sa307)</i> mutants, (G,H,I) <i>egl-9(sa307) drp-1(cq5)</i> double mutants, and (J,K,L) <i>egl-9(sa307) eat-3(ad426)</i> double mutants under conditions of (A,D,G,J) normoxia, (B,E,H,K) following 24 hours of anoxia, or (C,F,I,L) following 3 hours of reoxygenation post-anoxia. (M,N) Quantification of the mean (M) length and (N) number of mitochondria along the ventral cord for the indicated genotypes and conditions. Red bars indicate normoxia, blue bars indicate anoxia, and purple stippled bars indicate reoxygenation. ANOVA followed by Dunnett's multiple comparison to wild type, normoxia (***p<0.001, **p<0.01, *p<0.05). N = 15–35 animals per condition and/or genotype. Error bars indicate SEM. Bar, 5 µm.</p

STL-1 resides at mitochondria and is regulated by SKN-1.

<p>The fluorescence of (A,B) STL-1::GFP from a <i>P_glr-1::STL-1::GFP</i> transgene, and (C,D) TOM20::mCherry from a <i>P_glr-1::TOM20::mCherry</i> transgene, was observed in (A,C,E) command interneuron cell bodies, including PVC, and (B,D,F) ventral cord neurites of wild-type animals. (E,F) Merged images. The fluorescence of (G) STL-1::GFP from a <i>P_stl-1::STL-1::GFP</i> transgene was observed in the hypodermal epithelia of wild-type animals stained with (H) MitoTracker Red. (I) Merged image. (J) Mean levels of <i>stl-1</i> mRNA (relative to wild type) as measured by qRT-PCR in the indicated genotypes and conditions. Red bars indicate normoxia, blue bars indicate anoxia, and gray bars indicate paraquat treatment. Bar, 5 µm.</p

STL-1 is required for anoxia-induced mitochondrial hyperfusion.

<p>The fluorescence of MitoGFP was observed along ventral cord neurites of (A,B,C) wild-type animals, (D,E,F) <i>egl-9(sa307)</i> mutants, (G,H,I) <i>stl-1(tm1544)</i> mutants, and (J,K,L) <i>egl-9(sa307) stl-1(tm1544)</i> double mutants under conditions of (A,D,G,J) normoxia, (B,E,H,K) following 24 hours of anoxia, or (C,F,I,L) following 3 hours of reoxygenation post-anoxia. (M–P) Quantification of the mean (M,O,P) length and (N) number of mitochondria along the ventral cord for the indicated genotypes and conditions. (Q) Quantification of behavioral recovery (number of animals moving after 10 minutes of reoxygenation) of animals following 24 hours anoxia. Red bars indicate normoxia, blue bars indicate anoxia, and purple stippled bars indicate reoxygenation. ANOVA followed by Dunnett's multiple comparison to wild type, normoxia (***p<0.001), or by Bonferoni's multiple comparison test for the indicated comparison (#p<0.01). N = 15–35 animals per condition and/or genotype. Error bars indicate SEM. Bar, 5 µm.</p

The hypoxia response pathway regulates mitochondrial hyperfusion upon anoxia recovery.

<p>The fluorescence of MitoGFP was observed along ventral cord neurites of (A,B,C) wild-type animals, (D,E,F) <i>egl-9(sa307)</i> mutants, (G,H,I) <i>hif-1(ia4)</i> mutants, (J,K,L) <i>egl-9(sa307) hif-1(ia4)</i> double mutants, and (M,N,O) <i>egl-9</i> mutants with a transgene expressing the wild-type EGL-9A cDNA from the <i>glr-1</i> promoter. Conditions included (A,D,G,J,M) normoxia, (B,E,H,K,N) following 24 hours of anoxia, or (C,F,I,L,O) following 3 hours of reoxygenation post-anoxia. (P,Q) Quantification of the mean (P) length and (Q) number of mitochondria along the ventral cord for the indicated genotypes and conditions. (R) Quantification of behavioral recovery (number of animals moving after 10 minutes of reoxygenation) of animals following 24 hours anoxia. Red bars indicate normoxia, blue bars indicate anoxia, and purple stippled bars indicate reoxygenation. ANOVA followed by Dunnett's multiple comparison to wild type, normoxia (#p<0.001, **p<0.01, *p<0.05). N = 15–35 animals per condition and/or genotype. Error bars indicate SEM. Bar, 5 µm.</p