Identifying Roadkill Hotspots Using a Running Average

The identification of roadkill hotspots is necessary prior to the consideration of wildlife road mortality mitigation measures. In a previous study, 178 roadkill specimens were tallied via a driving survey along 21.4 km (13.3 mi) on three connected roadways in Baldwin County, Georgia. Roadkill locations were recorded to the nearest 0.16 km (0.1 mi) using the vehicle odometer. In the current study, location data were used to generate three graphical displays of roadkill distribution: 1) a linear graph of roadkills per 0.16 km (0.1 mi) bin; 2) a linear graph of roadkills per 0.8 km (0.5 mi) bin; and 3) a linear graph with a continuous running average incorporating 0.48 km (0.3 mi). The number and position of the peaks on each graph were compared in relation to roadway features that may influence animal movement and mortality such as vegetative boundaries, stream crossings, hills, and curves. The running average plot provided the best visual illustration of roadkill hotspot locations in relation to roadside features. The running average is a good technique to quickly and accurately identify hotspot locations and could help resource managers plan mitigation strategies to decrease wildlife road mortality

Novel Hypomorphic Alleles of the Mouse Tyrosinase Gene Induced by CRISPR-Cas9 Nucleases Cause Non-Albino Pigmentation Phenotypes

<div><p>Tyrosinase is a key enzyme in melanin biosynthesis. Mutations in the gene encoding tyrosinase (<i>Tyr</i>) cause oculocutaneous albinism (OCA1) in humans. Alleles of the <i>Tyr</i> gene have been useful in studying pigment biology and coat color formation. Over 100 different <i>Tyr</i> alleles have been reported in mice, of which ≈24% are spontaneous mutations, ≈60% are radiation-induced, and the remaining alleles were obtained by chemical mutagenesis and gene targeting. Therefore, most mutations were random and could not be predicted <i>a priori</i>. Using the CRISPR-Cas9 system, we targeted two distinct regions of exon 1 to induce pigmentation changes and used an <i>in vivo</i> visual phenotype along with heteroduplex mobility assays (HMA) as readouts of CRISPR-Cas9 activity. Most of the mutant alleles result in complete loss of tyrosinase activity leading to an albino phenotype. In this study, we describe two novel in-frame deletion alleles of <i>Tyr</i>, <i>dhoosara</i> (Sanskrit for gray) and <i>chandana</i> (Sanskrit for sandalwood). These alleles are hypomorphic and show lighter pigmentation phenotypes of the body and eyes. This study demonstrates the utility of CRISPR-Cas9 system in generating domain-specific in-frame deletions and helps gain further insights into structure-function of <i>Tyr</i> gene.</p></div

Details of the F0 animals obtained from CRISPR-Cas9 injections.

<p>Details of the F0 animals obtained from CRISPR-Cas9 injections.</p

CRISPR targeting and mutation detection by heteroduplex mobility assay in the <i>Tyr</i> gene.

<p>(A) Schematic showing CRISPR targeting regions (blue bold arrows), PCR primer binding sites, and amplicon sizes. (B) Breeding scheme with genotype of the zygotes used for CRISPR-Cas9 injections, and the CRISPR target sequences on both chromosomes. (C, D, E) Images of ethidium bromide stained polyacrylamide gels (6%) showing separation of homoduplex and heteroduplex PCR amplicons from CRISPR-Cas9 RNA injected, single cultured blastocysts (C, arrowheads) and tail DNA of potential founder mice (D, E). Gels on the left correspond to the 5’CRISPR target site, and those on the right correspond to the 3’CRISPR target site. Small and large square brackets indicate homoduplex and heteroduplex bands, respectively. L = 100 bp ladder; C = uninjected wildtype control.</p

Schematic of possible sequence of events producing related <i>dhoosara</i> and <i>chandana</i> alleles.

<p>CRISPR-Cas9 nuclease activity in the zygote at the 5’ target site on the wildtype chromosome results in a 15 bp deletion. After the cell division, nuclease activity persists and creates a DSB at the 3’ target site on the previously modified chromosome. NHEJ repair in this daughter cell results in a second, 3 bp deletion.</p

Multiples mutant alleles resulting from NHEJ at 5’ and 3’ CRISPR target sites.

<p>Multiples mutant alleles resulting from NHEJ at 5’ and 3’ CRISPR target sites.</p

Comparison of <i>dhoosara</i> and <i>chandana</i> with the albino and black/wild type mice.

<p>(A) Dorsal view showing the coat color of albino, <i>chandana</i>, <i>dhoosara</i>, and black animals. Increasing levels of pigmentation can be seen in the hind limbs and tips of tails as well. (B) Frontal view of the face with increasing pigmentation in the nose region. Differences in the eye color can be noted. (C) Brightfield images of RPE wholemounts from Albino, <i>chandana</i>, <i>dhoosara</i> and Black mice. (D) Brightfield images of retinal cryosections from Albino, <i>chandana</i>, <i>dhoosara</i> and black mice. The RPE layer is the only layer within the eyeball that contains pigment. Scale bar = 50 μm. (E) Mean gray values (quantified in ImageJ) obtained from the RPE wholemounts reflecting relative intensity of RPE cell pigmentation. Results are presented in a bar chart with standard error of mean used for error bars. All groups are significantly different from each other (p<0.05, t-test). (F) Western Blot of tyrosinase protein isolated from skin of albino (alb), <i>chandana (cha)</i>, <i>dhoosara (dho)</i>, and black animals (arrow points to the 60 kDa tyrosinase band); 50 kDa tubulin protein (lower panel) is used as a control.</p