Correction: CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants.

[This corrects the article DOI: 10.1371/journal.pone.0261331.].

Ultraviolet vision in anemonefish improves colour discrimination.

In many animals, ultraviolet (UV) vision guides navigation, foraging, and communication, but few studies have addressed the contribution of UV signals to colour vision, or measured UV discrimination thresholds using behavioural experiments. Here, we tested UV colour vision in an anemonefish (Amphiprion ocellaris) using a five-channel (RGB-V-UV) LED display. We first determined that the maximal sensitivity of the A. ocellaris UV cone was ∼386 nm using microspectrophotometry. Three additional cone spectral sensitivities had maxima at ∼497, 515 and ∼535 nm. We then behaviourally measured colour discrimination thresholds by training anemonefish to distinguish a coloured target pixel from grey distractor pixels of varying intensity. Thresholds were calculated for nine sets of colours with and without UV signals. Using a tetrachromatic vision model, we found that anemonefish were better (i.e. discrimination thresholds were lower) at discriminating colours when target pixels had higher UV chromatic contrast. These colours caused a greater stimulation of the UV cone relative to other cone types. These findings imply that a UV component of colour signals and cues improves their detectability, which likely increases the prominence of anemonefish body patterns for communication and the silhouette of zooplankton prey.

Short-wavelength-sensitive 1 (SWS1) opsin gene duplications and parallel visual pigment tuning support ultraviolet communication in damselfishes (Pomacentridae).

Damselfishes (Pomacentridae) are one of the most behaviourally diverse, colourful and species-rich reef fish families. One remarkable characteristic of damselfishes is their communication in ultraviolet (UV) light. Not only are they sensitive to UV, they are also prone to have UV-reflective colours and patterns enabling social signalling. Using more than 50 species, we aimed to uncover the evolutionary history of UV colour and UV vision in damselfishes. All damselfishes had UV-transmitting lenses, expressed the UV-sensitive SWS1 opsin gene, and most displayed UV-reflective patterns and colours. We find evidence for several tuning events across the radiation, and while SWS1 gene duplications are generally very rare among teleosts, our phylogenetic reconstructions uncovered two independent duplication events: one close to the base of the most species-rich clade in the subfamily Pomacentrinae, and one in a single Chromis species. Using amino acid comparisons, we found that known spectral tuning sites were altered several times in parallel across the damselfish radiation (through sequence change and duplication followed by sequence change), causing repeated shifts in peak spectral absorbance of around 10 nm. Pomacentrinae damselfishes expressed either one or both copies of SWS1, likely to further finetune UV-signal detection and differentiation. This highly advanced and modified UV vision among damselfishes, in particular the duplication of SWS1 among Pomacentrinae, might be seen as a key evolutionary innovation that facilitated the evolution of the exuberant variety of UV-reflectance traits and the diversification of this coral reef fish lineage.

Higher ultraviolet skin reflectance signals submissiveness in the anemonefish, Amphiprion akindynos.

Ultraviolet (UV) vision is widespread among teleost fishes, of which many exhibit UV skin colors for communication. However, aside from its role in mate selection, few studies have examined the information UV signaling conveys in other socio-behavioral contexts. Anemonefishes (subfamily, Amphiprioninae) live in a fascinating dominance hierarchy, in which a large female and male dominate over non-breeding subordinates, and body size is the primary cue for dominance. The iconic orange and white bars of anemonefishes are highly UV-reflective, and their color vision is well tuned to perceive the chromatic contrast of skin, which we show here decreases in the amount of UV reflectance with increasing social rank. To test the function of their UV-skin signals, we compared the outcomes of staged contests over dominance between size-matched Barrier Reef anemonefish (Amphiprion akindynos) in aquarium chambers viewed under different UV-absorbing filters. Fish under UV-blocking filters were more likely to win contests, where fish under no-filter or neutral-density filter were more likely to submit. For contests between fish in no-filter and neutral density filter treatments, light treatment had no effect on contest outcome (win/lose). We also show that sub-adults were more aggressive toward smaller juveniles placed under a UV filter than a neutral density filter. Taken together, our results show that UV reflectance or UV contrast in anemonefish can modulate aggression and encode dominant and submissive cues, when changes in overall intensity are controlled for.

CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants.

Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of zygotes was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR/Cas9 in A. ocellaris, as a platform for studying gene function in anemonefishes and other reef fishes.

Molecular Evolution of Ultraviolet Visual Opsins and Spectral Tuning of Photoreceptors in Anemonefishes (Amphiprioninae).

Many animals including birds, reptiles, insects, and teleost fishes can see ultraviolet (UV) light (shorter than 400 nm), which has functional importance for foraging and communication. For coral reef fishes, shallow reef environments transmit a broad spectrum of light, rich in UV, driving the evolution of diverse spectral sensitivities. However, the identities and sites of the specific visual genes that underly vision in reef fishes remain elusive and are useful in determining how evolution has tuned vision to suit life on the reef. We investigated the visual systems of 11 anemonefish (Amphiprioninae) species, specifically probing for the molecular pathways that facilitate UV-sensitivity. Searching the genomes of anemonefishes, we identified a total of eight functional opsin genes from all five vertebrate visual opsin subfamilies. We found rare instances of teleost UV-sensitive SWS1 opsin gene duplications that produced two functionally coding paralogs (SWS1α and SWS1ß) and a pseudogene. We also found separate green sensitive RH2A opsin gene duplicates not yet reported in the family Pomacentridae. Transcriptome analysis revealed false clown anemonefish (Amphiprion ocellaris) expressed one rod opsin (RH1) and six cone opsins (SWS1ß, SWS2B, RH2B, RH2A-1, RH2A-2, LWS) in the retina. Fluorescent in situ hybridization highlighted the (co-)expression of SWS1ß with SWS2B in single cones, and either RH2B, RH2A, or RH2A together with LWS in different members of double cone photoreceptors (two single cones fused together). Our study provides the first in-depth characterization of visual opsin genes found in anemonefishes and provides a useful basis for the further study of UV-vision in reef fishes.

More than noise: context-dependent luminance contrast discrimination in a coral reef fish (Rhinecanthus aculeatus).

Achromatic (luminance) vision is used by animals to perceive motion, pattern, space and texture. Luminance contrast sensitivity thresholds are often poorly characterised for individual species and are applied across a diverse range of perceptual contexts using over-simplified assumptions of an animal's visual system. Such thresholds are often estimated using the receptor noise limited model (RNL). However, the suitability of the RNL model to describe luminance contrast perception remains poorly tested. Here, we investigated context-dependent luminance discrimination using triggerfish (Rhinecanthus aculeatus) presented with large achromatic stimuli (spots) against uniform achromatic backgrounds of varying absolute and relative contrasts. 'Dark' and 'bright' spots were presented against relatively dark and bright backgrounds. We found significant differences in luminance discrimination thresholds across treatments. When measured using Michelson contrast, thresholds for bright spots on a bright background were significantly higher than for other scenarios, and the lowest threshold was found when dark spots were presented on dark backgrounds. Thresholds expressed in Weber contrast revealed lower thresholds for spots darker than their backgrounds, which is consistent with the literature. The RNL model was unable to estimate threshold scaling across scenarios as predicted by the Weber-Fechner law, highlighting limitations in the current use of the RNL model to quantify luminance contrast perception. Our study confirms that luminance contrast discrimination thresholds are context dependent and should therefore be interpreted with caution.

Visual system diversity in coral reef fishes.

Coral reefs are one of the most species rich and colourful habitats on earth and for many coral reef teleosts, vision is central to their survival and reproduction. The diversity of reef fish visual systems arises from variations in ocular and retinal anatomy, neural processing and, perhaps most easily revealed by, the peak spectral absorbance of visual pigments. This review examines the interplay between retinal morphology and light environment across a number of reef fish species, but mainly focusses on visual adaptations at the molecular level (i.e. visual pigment structure). Generally, visual pigments tend to match the overall light environment or micro-habitat, with fish inhabiting greener, inshore waters possessing longer wavelength-shifted visual pigments than open water blue-shifted species. In marine fishes, particularly those that live on the reef, most species have between two (likely dichromatic) to four (possible tetrachromatic) cone spectral sensitivities and a single rod for crepuscular vision; however, most are trichromatic with three spectral sensitivities. In addition to variation in spectral sensitivity number, spectral placement of the absorbance maximum (λmax) also has a surprising degree of variability. Variation in ocular and retinal anatomy is also observed at several levels in reef fishes but is best represented by differences in arrangement, density and distribution of neural cell types across the retina (i.e. retinal topography). Here, we focus on the seven reef fish families most comprehensively studied to date to examine and compare how behaviour, environment, activity period, ontogeny and phylogeny might interact to generate the exceptional diversity in visual system design that we observe.

Triggerfish uses chromaticity and lightness for object segregation.

Humans group components of visual patterns according to their colour, and perceive colours separately from shape. This property of human visual perception is the basis behind the Ishihara test for colour deficiency, where an observer is asked to detect a pattern made up of dots of similar colour with variable lightness against a background of dots made from different colour(s) and lightness. To find out if fish use colour for object segregation in a similar manner to humans, we used stimuli inspired by the Ishihara test. Triggerfish (Rhinecanthus aculeatus) were trained to detect a cross constructed from similarly coloured dots against various backgrounds. Fish detected this cross even when it was camouflaged using either achromatic or chromatic noise, but fish relied more on chromatic cues for shape segregation. It remains unknown whether fish may switch to rely primarily on achromatic cues in scenarios where target objects have higher achromatic contrast and lower chromatic contrast. Fish were also able to generalize between stimuli of different colours, suggesting that colour and shape are processed by fish independently.

Lessons learned from Hurricane Ike.

After the poorly planned evacuation for Hurricane Rita in 2005, many health care systems in the Houston area updated the disaster plans they would implement in the event of a major disaster. In September 2008, Texas health care systems in the Houston-Galveston area had the opportunity to execute those plans when Hurricane Ike made landfall. Despite hours of hurricane preparation at the Texas Orthopedic Hospital in Houston, TX, before the storm, we found that there were still lessons to be learned from Hurricane Ike that can be used by other health care systems to prepare for disaster.

The transition from open to endoscopic saphenous vein harvesting and its clinical impact: The Texas Heart Institute experience.

Open saphenous vein harvesting can be associated with wound complications, incision pain, prolonged convalescence, and poor cosmetic results. Endoscopic vein harvesting has been widely used for prevention of these problems. We compared outcomes of open and endoscopic vein harvesting for coronary artery bypass grafting at the Texas Heart Institute. We retrospectively analyzed data from 1,573 consecutive coronary artery bypass procedures performed at our institution during a 20-month period. Each procedure included saphenectomy by endoscopic vein harvesting (n = 588) performed by physician assistants, or by traditional open vein harvesting (n = 985) performed by physicians or physician assistants. The primary outcome variable was the incidence of postoperative leg infections. Both groups were similar in terms of preoperative risk factors. After surgery, leg wound infections were significantly less frequent in the endoscopic vein harvesting group (3/588, 0.5%) than in the open vein harvesting group (27/985, 2.7%; P < 0.002). The most common organism involved in leg infections was Staphylococcus (20/30, 66%): S. aureus was present in 14 of 30 infections (47%). Open vein harvesting was the only significant independent risk factor for leg infection. We conclude that endoscopic vein harvesting reduces leg wound infections, is safe and reliable, and should be the standard of care when venous conduits are required for coronary artery bypass grafting and vascular procedures. Although the transition from open to endoscopic vein harvesting can be challenging in institutions, it can be successful if operators receive adequate training in endoscopic technique and are supported by surgeons and staff.

5-fluorouracil forward mutation assay in Salmonella: determination of mutational target and spontaneous mutational spectra.

A forward mutation assay in Salmonella typhimurium that selects for 5-fluoruracil (FU) resistance has been developed. The two genes possibly involved in FU resistance, the uracil phosphoribosyl transferase gene (upp) and the uracil transport protein (uraA), have been cloned from S. typhimurium and sequenced. One hundred percent of FU-resistant clones display sequence changes in the upp gene, indicating that its loss is the major mechanism involved in FU resistance. The spontaneous mutational spectra at the upp locus were then determined in two S. typhimurium strains, FU100 and FU1535, that differ only in the presence of pKM101 plasmid. The pKM101 plasmid provides error-prone replicative bypass of DNA lesions and renders FU100 more susceptible to induced mutagenesis. Fluctuation analysis of FU-resistant clones demonstrated a 10-fold higher spontaneous mutation rate at the upp locus in FU100 relative to FU1535. Over 300 independent FU-resistant clones were then used to generate the spectra at the upp locus in both the strains. Approximately 40% of all the mutations were base substitutions, present at the same relative percentage in both the strains. Frameshift mutations also accounted for approximately 40% of the total; however, their incidence was slightly elevated in FU100. The remaining mutations were larger insertions and deletions, which were both slightly elevated in FU1535. pKM101 significantly elevated the rate of all classes of mutations at the upp locus, with profound effects on A:T to T:A transversions and -2-base frameshift mutations. These initial mutational spectra at the upp locus reveal 147 mutable sites, or 23% of the total 627-base coding sequence and suggest that the target can detect a diverse spectrum of mutagenic events.

Establishing a collaborative relationship with a college of nursing.

Perioperative nursing typically is a small part of nursing school curricula, which makes it difficult to recruit new graduates into the perioperative field. St Luke's Episcopal Hospital, Houston, has taken a unique approach to addressing this problem by establishing a collaborative relationship with Prairie View A&M University, Houston. This collaborative relationship introduces perioperative nursing to nursing students who may never have considered the OR as an area of practice. In addition, it provides an opportunity for students to network with perioperative nurses, and it serves as a method for recruiting new graduates to St Luke's Episcopal Hospital.

Volatile hydrocarbon emissions from a diesel fuel-contaminated soil bioremediation facility.

Concerns have been expressed that emissions of volatile hydrocarbons (HCs) from bioremediation facilities containing soils contaminated with petroleum HCs may negatively impact regional air quality or human health. Little information is available regarding the emission of HCs from bioremediation sites, and few field studies have been performed during which the flux of HCs has been directly measured during bioremediation. To aid in answering questions about the impact of bioremediation facilities on the atmospheric environment, a two-part field study was conducted over summer 1996 at a remote landfarm in northern Ontario where diesel fuel-contaminated soil was undergoing bioremediation. Volatile total hydrocarbon (THC) atmospheric flux measurements were successfully taken over 18 days using a flux gradient micrometeorological technique incorporating a THC detector constructed in-house. Peak THC emissions reached 131 microg C/m2/sec shortly after implementation and tilling of the landfarm soil. The influence of soil temperature and tillage on THC emissions was examined. Off-site inhalation exposure was considered with the aid of an areal source model and results from speciated air samples collected on sorbent tubes and analyzed via gas chromatography/mass spectrometry (GCMS) techniques.