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One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research., (© 2024. The Author(s).)

Franz Kafka had beautiful eyes. So striking, that many of the famous author's friends and peers commented on them - but quite variously ('dark', 'brown', 'grey' & 'blue'). Eye colour as perceived by an observer is subjective, being influenced by physiological, environmental, and even sociocultural factors. In a policing context, this does not mean that trait information such as eye colour is not valuable (far from it), but that it must be managed carefully. The Australian Federal Police has recently implemented a forensic DNA phenotyping (FDP, aka. physical trait prediction or PTP) capability, utilising massively parallel sequencing DNA technology to predict an individual's eye colour, biogeographical ancestry and sex from a crime scene sample. This information alone is not itself 'intelligence', but can be used to generate intelligence through holistic analyses undertaken within a transdisciplinary, all-source forensic intelligence (FORINT) framework. FORINT outputs posit abductive propositions typically at the activity/offence level, to provide insight and influence decision making. However, the use of predicted traits requires that they are compared to something; all Australian police databases include fields for physical traits, but no uniform standard is applied across all agencies. Moreover, collection is inconsistent and no automated systems are in place to capture such data systematically. Consider the 'Kafka problem': his peers gave multiply divergent descriptions of his eyes. If a Biology unit had predicted the eye colour of an 'unidentified author' using DNA - how would Kafka be confidently nominated as the contributor? We posit three maxims for law enforcement: (1) To expand the operational utility of forensic science in line with police demands, forensic science should operationalise FDP (e.g. operationally to rank a list of persons of interest, focus lines of enquiry in serious & organised crime, or assist with human remains identification). (2) Such advanced biological techniques are best delivered through an all-source FORINT framework, to maximise opportunities and minimise risk. (3) One cannot pursue techno-scientific advancements in isolation; it is also necessary to influence the operational posture for their implementation. In this paper we explore these issues and provide recommendations relating to (a) police practices, (b) image capture systems, and (c) research opportunities. Phenotypic trait prediction has great potential and can be operationalised effectively through a rigorous FORINT framework. However, there is (continual) work to be done to enhance the operational capabilities that are complementary to - but necessary for - effective forensic science contribution to investigations., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: This study assesses the accuracy of the IrisPlex system, a genetic eye color prediction tool for forensic analysis, in the Kazakh population. The study compares previously published genotypes of 515 Kazakh individuals from varied geographical and ethnohistorical contexts with phenotypic data on their eye color, introduced for the first time in this research., Results: The IrisPlex panel's effectiveness in predicting eye color in the Kazakh population was validated. It exhibited slightly lower accuracy than in Western European populations but was higher than in Siberian populations. The sensitivity was notably high for brown-eyed individuals (0.99), but further research is needed for blue and intermediate eye colors. This study establishes IrisPlex as a useful predictive tool in the Kazakh population and provides a basis for future investigations into the genetic basis of phenotypic variations in this diverse population., (© 2024. The Author(s).)

Purpose: To determine the correlation of Fitzpatrick Skin Type (FST) and iris color with tumor size (tumor thickness and basal diameter) in patients with uveal melanoma., Design: Retrospective Cohort METHODS: Retrospective cohort from a single ocular oncology center of 823 patients with uveal melanoma and documented FST, iris color, and tumor size. Patients were classified by FST (type I, II, and III-V) and iris color (blue, green, and brown) on the basis of external facial photography. There were no FST type VI patients. Tumor thickness was classified into small [< 3 millimeter (mm)], medium (3.1-8.0 mm), or large (> 8.0 mm), and basal diameter into small (< 10 mm), medium (10.1-15 mm) or large (> 15 mm). The correlation of FST and iris color with tumor thickness and basal diameter was evaluated using the Kruskal-Wallis H test., Results: The FST classification was type I (n = 92, 11%), type II (n = 643, 78%), or III-V (n = 88, 11%), and iris color was blue (n = 472, 57%), green (n = 102, 12%), or brown (n = 249, 30%). A comparison of FST revealed differences in mean tumor thickness (P = 0.04) and basal diameter (P = 0.006). Iris color showed no difference for mean tumor thickness (P = 0.41) or basal diameter (P = 0.48). There was a statistically significant difference with brown iris color relative to FST III-V for mean tumor thickness (P = 0.003) and basal diameter (P = 0.001) but no difference with blue or green iris color (P > 0.05)., Conclusions: Iris color alone showed no difference in tumor size, but those with brown iris color and FST type III-V demonstrated larger tumor thickness and basal diameter., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Understanding the iris colour might be a useful biomarker in predicting emotional health status. We aimed to examine the relationship of iris colour with subjective emotional status in a sample of Chinese adolescents aged 13 to 14 years., Methods: A total of 2346 students from ten middle schools in Mojiang participated the examination. The emotional health status was measured by the adolescent self-reported PedsQL 4.0. We obtained standardized slit-lamp photographs and developed a grading system assessing iris colour (higher grade denoting darker). Logistic regression models were fitted to assess the relationship between iris colour and emotional status., Results: The trend of decreasing negative emotional status with darker iris colour was significantly. Compared with grades 1 and 2 of iris colour, the students with grades 4 and 5 had less negative emotional status (OR fear  = 0.56, 95 % CI: 0.41, 0.76; OR sadness  = 0.67, 95 % CI: 0.49, 0.92; OR anger  = 0.71, 95 % CI: 0.52, 0.98). In addition, the trend did not change after adjusting for potential confounders, including height, waist circumstance, sleep habits and computer times in logistic regression models., Limitations: Only negative emotional indexes were selected as evaluative materials, it is unclear whether the pattern found in the present study also exists with regard to positive emotional indexes, such as happiness., Conclusion: Darker iris colour might be associated with less negative emotional status in Chinese adolescents., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Phenotypic trait prediction in ancient DNA analysis can provide information about the external appearance of individuals from past human populations. Some studies predicting eye and hair color in ancient adult skeletons have been published, but not for ancient subadult skeletons, which are more prone to decay. In this study, eye and hair color were predicted for an early medieval adult skeleton and a subadult skeleton that was anthropologically characterized as a middle-aged man and a subadult of unknown sex about 6 years old. When processing the petrous bones, precautions were taken to prevent contamination with modern DNA. The MillMix tissue homogenizer was used for grinding, 0.5 g of bone powder was decalcified, and DNA was purified in Biorobot EZ1. The PowerQuant System was used for quantification and a customized version of the HIrisPlex panel for massive parallel sequencing (MPS) analysis. Library preparation and templating were performed on the HID Ion Chef Instrument and sequencing on the Ion GeneStudio S5 System. Up to 21 ng DNA/g of powder was obtained from ancient petrous bones. Clean negative controls and no matches with elimination database profiles confirmed no contamination issue. Brown eyes and dark brown or black hair were predicted for the adult skeleton and blue eyes and brown or dark brown hair for the subadult skeleton. The MPS analysis results obtained proved that it is possible to predict hair and eye color not only for an adult from the Early Middle Ages, but also for a subadult skeleton dating to this period., (© 2023. The Author(s).)

Human ear morphology prediction with SNP-based genotypes is growing in forensic DNA phenotyping and is scarcely explored in Pakistan as a part of EVCs (externally visible characteristics). The ear morphology prediction assays with 21 SNPs were assessed for their potential utility in forensic identification of population. The SNaPshot™ multiplex chemistries, capillary electrophoresis methods and GeneMapper™ software were used for obtaining genotypic data. A total of 33 ear phenotypes were categorized with digital photographs of 300 volunteers. SHEsis software was applied to make LD plot. Ordinal and multinomial logistic regression was implemented for association testing. Multinomial logistic regression was executed to construct the prediction model in 90% training and 10% testing subjects. Several influential SNPs for ear phenotypic variation were found in association testing. The model based on genetic markers predicted ear phenotypes with moderate to good predictive accuracies demonstrated with the area under curve (AUC), sensitivity and specificity of predicted phenotypes. As an additional EVC, the estimated ear phenotypic profiles have the possibility of determining the human ear morphology differences in unknown biological samples found in crimes that do not result in a criminal database hit. Furthermore, this can help in facial reconstruction and act as an investigational lead., (© 2022. The Author(s).)

Competing Interests: The authors report no conflicts of interest.

In recent decades, the use of genetic polymorphisms related to specific phenotypes, such as eye color, has greatly contributed to the development of the research field called forensic DNA phenotyping (FDP), enabling the investigators of crime cases to reduce the number of suspects, making their work faster and more precise. Eye color is a polygenic phenotype, and many genetic variants have been highlighted, with the major contributor being the HERC2-OCA2 locus, where many single nucleotide variations (SNPs) were identified. Interestingly, the HERC2-OCA2 locus, containing the intronic SNP rs12913832, the major eye color determinant, shows a high level of evolutionary conservation across many species of vertebrates. Currently, there are some genetic panels to predict eye color by genomic DNA analysis, even if the exact role of the SNP variants in the formation of eye color is still poorly understood, with a low level of predictivity in the so-called intermediate eye color. Many variants in OCA2 , HERC2 , and other genes lie in introns or correspond to synonymous variants, highlighting greater complexity in the mechanism of action of such genes than a simple missense variation. Here, we show the main genes involved in oculocutaneous pigmentation and their structural and functional features, as well as which genetic variants show the highest level of eye color predictivity in currently used FDP assays. Despite the great recent advances and impact of FDP in criminal cases, it is necessary to enhance scientific research to better understand the mechanism of action behind each genetic variant involved in eye color, with the goal of obtaining higher levels of prediction.

Ancient DNA analyses help to solve the problems related to the genogeographic origin and migration patterns of populations. The Khazar Khaganate is a subject of controversy among researchers. Its complex historical development, lack of a sufficient number of artistic and written sources, the disappearance of representatives of Khazar culture leaves open the question of the appearance of the Khazars. DNA phenotyping of bone remains from elite burials of the Khazar period of Southern Russia was carried out with respect to eye color, hair color, skin color, and AB0 blood groups. Eight out of 10 individuals had brown eyes, dark hair (to varying degrees), and a predominantly dark skin during their lifetime. Individuals from two burials had gray-blue eyes, and one individual had blond hair. The most probable AB0 blood group was identified in eight people, of which five blood group 0 (I) group, four had blood group A (II), and one had blood group B (III). The allele frequency distribution was assessed for ten population-specific autosomal markers and suggested high heterogeneity for the ethnogeographic origin of the Khazars examined. The results are evidence for ethnocultural, genetic, and phenotypic diversity of the Khazar Khaganate.

Insects display a vast array of eye and body colors. Genes encoding products involved in biosynthesis and deposition of pigments are ideal genetic markers, contributing, for example, to the power of Drosophila genetics. Oncopeltus fasciatus is an emerging model for hemimetabolous insects, a member of the piercing-sucking feeding order Hemiptera, that includes pests and disease vectors. To identify candidate visible markers for O. fasciatus, we used parental and nymphal RNAi to identify genes that altered eye or body color while having no deleterious effects on viability. We selected Of-vermilion for CRISPR/Cas9 genome editing, generating three independent loss-of-function mutant lines. These studies mapped Of-vermilion to the X-chromosome, the first assignment of a gene to a chromosome in this species. Of-vermilion homozygotes have bright red, rather than black, eyes and are fully viable and fertile. We used these mutants to verify a role for Of-xdh1, ortholog of Drosophila rosy, in contributing to red pigmentation using RNAi. Rather than wild-type-like red bodies, bugs lacking both vermilion and xdh1 have bright yellow bodies, suggesting that ommochromes and pteridines contribute to O. fasciatus body color. Our studies generated the first gene-based visible marker for O. fasciatus and expanded the genetic toolkit for this model system., (© 2023. The Author(s).)

Studies have indicated that people are attracted to partners who resemble themselves or their parents, in terms of physical traits including eye color. We might anticipate this inclination to be relatively stable, giving rise to a sequential selection of similar partners who then represent an individual's "type". We tested this idea by examining whether people's sequential partners resembled each other at the level of eye color. We gathered details of the eye colors of the partners of participants (N = 579) across their adult romantic history (N = 3250 relationships), in three samples, comprising two samples which made use of self-reports from predominantly UK-based participants, and one which made use of publicly available information about celebrity relationship histories. Recorded partner eye colors comprised black (N = 39 partners), dark brown (N = 884), light brown (N = 393), hazel (N = 224), blue (N = 936), blue green (N = 245), grey (N = 34), and green (N = 229). We calculated the proportion of identical eye colors within each participant's relationship history, and compared that to 100,000 random permutations of our dataset, using t-tests to investigate if the eye color of partners across an individual's relationship history was biased relative to chance (i.e., if there was greater consistency, represented by higher calculated proportions of identical eye colors, in the original dataset than in the permutations). To account for possible eye color reporting errors and ethnic group matching, we ran the analyses restricted to White participants and to high-confidence eye color data; we then ran the analyses again in relation to the complete dataset. We found some limited evidence for some consistency of eye color across people's relationship histories in some of the samples only when using the complete dataset. We discuss the issues of small effect sizes, partner-report bias, and ethnic group matching in investigating partner consistency across time., (© 2022. The Author(s).)

Eye color prediction based on an individual's genetic information is of interest in the field of forensic genetics. In recent years, researchers have studied different genes and markers associated with this externally visible characteristic and have developed methods for its prediction. The IrisPlex represents a validated tool for homogeneous populations, though its applicability in populations of mixed ancestry is limited, mainly regarding the prediction of intermediate eye colors. With the aim of validating the applicability of this system in an admixed population from Argentina (n = 302), we analyzed the six single nucleotide variants used in that multiplex for eye color and four additional SNPs, and evaluated its prediction ability. We also performed a genotype-phenotype association analysis. This system proved to be useful when dealing with the extreme ends of the eye color spectrum (blue and brown) but presented difficulties in determining the intermediate phenotypes (green), which were found in a large proportion of our population. We concluded that these genetic tools should be used with caution in admixed populations and that more studies are required in order to improve the prediction of intermediate phenotypes., (© 2022 John Wiley & Sons Ltd/University College London.)

External eye appearance across primate species is diverse in shape and colouration, yet we still lack an explanation for the drivers of such diversity. Here we quantify substantial interspecific variation in eye shape and colouration across 77 primate species representing all extant genera of anthropoid primates. We reassess a series of hypotheses aiming to explain ocular variation in horizontal elongation and in colouration across species. Heavier body weight and terrestrial locomotion are associated with elongated eye outlines. Species living closer to the equator present more pigmented conjunctivae, suggesting photoprotective functions. Irises become bluer in species living further away from the equator, adding to existing literature supporting a circadian clock function for bluer irises. These results shift the current focus from communicative, to ecological factors in driving variation in external eye appearance in anthropoid primates. They also highlight the possibility that similar ecological factors contributed to selection for blue eyes in ancestral human populations living in northern latitudes., (© 2022. The Author(s).)

This paper presents a method for genotyping a panel of 60 single nucleotide polymorphisms (SNPs) using single-stage PCR followed by hybridization on a hydrogel biochip. The pool of analyzed polymorphisms consists of 41 SNPs included in the HIrisPlex-S panel, 4 SNPs of the AB0 gene (261G>Del, 297A>G, 657C>T, 681G>A), markers of the AMELX and AMELY genes, and 14 SNP markers of the Y chromosome haplogroups: B (M60), C (M130), D (CTS3946), E (M5388), G (P257), H (M2920), I (U179), J (M304), L (M185), N (M231), O (M175), Q (M1105), R (P224) and T (M272). These genetic data allow one to predict the phenotype of the desired person according to the characteristics of eye, hair, skin color, AB0 blood group, sex, and genogeographic origin in the male line. The setting protocol is simplified as much as possible to facilitate the introduction of the method into practice. The distribution of allele frequencies of the studied polymorphisms, as well as AB0 blood groups among the Slavs (N = 482), originating mainly from central Russia, was established.

This study aims to present the current state of knowledge on the DNA-based prediction of human externally visible characteristics of an unknown person based on the crime scene biological material left behind. This DNA sample is referred to as a “biological witness” and the procedure itself is called forensic DNA phenotyping (FDP). The analytic part of this work is based on scholarly articles published between 2015 and 2021. The electronic search of relevant references was conducted according to the PRISMA methodology in March 2021 at EBSCO Discovery Service (EDS) at the Adam Mickiewicz University library and Google Scholar. The molecular basis of FDP, DNA markers used to predict sex, age, biogeographic origin and externally visible traits such as pigmentation (skin, eye and hair colour), hair morphology, facial morphology, presence of freckles, body height, body weight (obesity), male pattern baldness and myopia were described. Furthermore, methodological difficulties resulting from the polygenic inheritance of the studied traits, as well as social and ethical problems accompanying forensic DNA phenotyping were discussed. Finally, key themes for future research related to forensic DNA phenotyping were outlined.

Competing Interests: Competing interests: None declared.

Men with light eyes lack the dominant gene allele that codes for dark-brown eyes. Pairing with a woman who lacks the same allele must increase paternity confidence in these men, because any children with dark eyes would be extremely unlikely to have been fathered by them. This notion implies that men with light (blue or green) eyes should (1) prefer light-eyed women, especially in a long-term context, and (2) feel more threatened by light-eyed than by dark-eyed rivals. Yet because choosiness is costly and paternity concerns are entirely driven by the prospect of paternal investment, any such inclinations would be adaptive only in men who expect to invest in their children. Here I test these ideas using the data of over 1000 men who rated the facial attractiveness of potential partners, and the threat of potential rivals, whose eye color had been manipulated. Light-eyed men liked light-eyed women better (particularly as long-term companions), and feared light-eyed rivals more, than did dark-eyed men. An exploratory analysis showed that these large, robust effects disappeared in men who had felt rejected by their fathers while growing up-suggesting that such men are not expecting to invest in their own children either., (© 2021. The Author(s).)

It has been advocated before that appearance prediction of unknown suspects from crime scene DNA, in the context of Forensic DNA Phenotyping (FDP), is mostly suitable for single source DNA samples, whereas FDP from DNA mixtures to which more than one person contributed, is viewed challenging. With this report on a murder case, we practically demonstrate the feasibility of appearance DNA prediction of an unknown suspect from a mixed crime scene trace, to which the unknown suspect and the known victim had contributed. From this two-person DNA mixture, we successfully predicted eye, hair and skin color of the unknown suspect with the HIrisPlex-S system by applying targeted massively parallel sequencing (MPS). We argue that at least three factors benefit appearance DNA prediction of unknown suspects from mixed crime scene traces, which were met in this murder case: i) SNP genotype knowledge from reference DNA analysis for one of the two persons in the mixture (here the known victim), ii) about equal DNA contributions by both donors to the mixed crime scene stain, and iii) the use of MPS allowing quantitative SNP analysis. Moreover, we show that additionally analyzing animal DNA in this mixed crime scene trace provides further investigative information. We envision that the investigative DNA strategy that we applied here for analyzing a two-person mixed crime scene trace in a murder case, will be applied in the future to more criminal cases with two-person DNA mixtures, for instance sexual assault cases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To investigate the effects of pharmacological accommodation and cycloplegia on ocular measurements., Methods: Thirty-three healthy subjects [mean (±SD) age, 32.97 (±5.21) years] volunteered to participate in the study. Measurement of the axial length, macular and choroidal thickness, refractive error, and corneal topography, as well as anterior segment imaging, were performed. After these procedures, pharmacological accommodation was induced by applying pilocarpine eye drops (pilocarpine hydrochloride 2%), and the measurements were repeated. The measurements were repeated again after full cycloplegia was induced using cyclopentolate eye drops (cyclopentolate hydrochloride 1%). The correlations between the measurements were evaluated., Results: A significant increase in subfoveal choroidal thickness after applying 2% pilocarpine was identified (without the drops, 319.36 ± 90.08 µm; with pilocarpine instillation, 341.60 ± 99.19 µm; with cyclopentolate instillation, 318.36 ± 103.0 µm; p<0.001). A significant increase in the axial length was also detected (without the drops, 23.26 ± 0.83 mm; with pilocarpine instillation, 23.29 ± 0.84 mm; with cyclopentolate instillation, 23.27 ± 0.84 mm; p=0.003). Comparing pharmacological accommodation and cycloplegia revealed a significant difference in central macular thickness (with pilocarpine instillation, 262.27 ± 19.34 µm; with cyclopentolate instillation, 265.93 ± 17.91 µm; p=0.016). Pilocarpine-related miosis (p<0.001) and myopic shift (p<0.001) were more severe in blue eyes vs. brown eyes., Conclusion: Pharmacological accommodation may change ocular measurements, such as choroidal thickness and axial length. This condition should be considered when performing ocular measurements, such as intraocular lens power calculations.

Objective: Phacoemulsification is the most common cataract surgery that needs optimum circumstances in the field of surgery. This comparative pre- and postoperative study assessed the efficacy and safety of using adrenaline in the irrigating solution as an adjunct to preoperative topical mydriatics in dark irides during Phaco surgery., Patients and Methods: This was a prospective observational study that enrolled 421 cataract patients (421 eyes) with dark irides, who were scheduled for Phaco surgery from January 2019 to August 2020. All patients received intraoperative irrigation of a balanced salt solution containing adrenaline. The pulse rate and systolic and diastolic blood pressure of all patients were recorded pre- and postoperatively. In addition, the presence of intraoperative floppy-iris syndrome (IFIS), need for pupil mechanical dilatation, and incidence of posterior capsular rupture were recorded., Results: The sample consisted of 421 patients (421 eyes) all had dark irides. Pulse rate and systolic and diastolic blood pressure did not significantly increase post-operatively (p <0.001). Mechanical dilatation of the pupil was performed in one patient (0.24%) and seven eyes (1.66%) were found to have IFIS. There was no case of posterior capsule rupture., Conclusions: In comparison with the use of preoperative topical mydriatics alone, adding intracameral adrenaline to the irrigation fluid maintains better pupillary dilatation throughout Phacoemulsification surgery, thereby providing better clinical outcomes in dark irides, even in those with IFIS. Its use has no incremental effect on either blood pressure or pulse rate.

Introduction: Brown or dark brown eyes make it difficult to distinguish the contrast between a black pupil and the surrounding iris, which may result in clinical assessment errors. The pupillometer can be used to derive an indexed value, the Neurological Pupil index™ (NPi) for pupillary light reflex. However, there are limited data associating the NPi and iris colour. We examine the NPi and eye colour association., Methods: Data were pooled from the Establishing Normative Data for Pupillometer Assessments in Neuroscience Intensive Care (END-PANIC) Registry. The analysis includes 14,168 observations collected from 865 patients with neurological conditions who were admitted to the intensive care unit. Summary statistics and statistical models were developed to examine the association using Statistical Analysis Software (SAS) summary procedure., Results: The mean age of the cohort was 56 years (standard deviation = 17). Eye colour included dark brown (n = 339), blue (n = 234), brown (n = 173), green (n = 82), and other (n = 37). There was significant differences (p < 0.0001) between mean NPi values by eye colour [blue = 4.08 (0.92), brown = 3.34 (1.45), dark = 3.71 (1.33), green = 4.08 (0.67), other = 3.76 (1.25)]. However, a further random-effects mixed model after controlling for confounding variables revealed no significant difference in NPi values among different eye colour groups., Conclusions: The pupillary light reflex, when assessed using the pupillometer, is not dependent on the eye colour. Practitioners are not required to consider eye colour as a confounder when they perform pupillary assessment for examining patients with neurological conditions., (Copyright © 2019 Australian College of Critical Care Nurses Ltd. Published by Elsevier Ltd. All rights reserved.)

Ommochromes are the major pigments found in the eyes, eggs, wings and epidermis of insects. Here, we report the identification and characterization of the gene responsible for red-1 locus of Tribolium, whose mutants have white eyes due to lack of ommochrome pigments in the eyes. Using a candidate gene approach, we demonstrated that red-1 and peach mutants have molecular defects in the cardinal gene, which encodes a haem peroxidase that is considered to convert 3-hydroxykynurenine into ommochromes in pigment granules. Our experiments showed that the expression pattern of cardinal correlates well with the progression of eye pigmentation during pupal stages. We performed gene editing experiments using the Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control technique to disrupt the cardinal gene by adult injection, and were able to establish a novel cardinal mutant line. Our complementation test provided definitive genetic evidence that cardinal is located at the red-1 locus. The present study will lead to a greater understanding of the function and diversity of ommochrome pathway genes in insects. Our successful use of ReMOT Control in beetles will facilitate the development of more efficient and versatile systems for insect genome editing by simple adult injection., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Understanding the adaptive function of conspicuous coloration has been a major focus of evolutionary biology for much of the last century. Although considerable progress has been made in explaining how conspicuous coloration can be used in functions as diverse as sexual and social signaling, startling predators, and advertising toxicity [1], there remain a multitude of species that display conspicuous coloration that cannot be explained by existing theory. Here we detail a new "matador-like" divertive antipredator strategy based on conspicuous coloration in Trinidadian guppies (Poecilia reticulata). Guppies encountering predatory fish rapidly enhance the conspicuousness of their eyes by blackening their irises. By pitting biomimetic robotic guppies against real predatory fish, we show this conspicuous eye coloration diverts attacks away from the guppies' center of mass to their head. To determine the function of this seemingly counterintuitive behavior, we developed a method for simulating escape probabilities when live prey interact with ballistic attacking predators, and find this diversion effect significantly benefits black-eyed guppies because they evade capture by rapidly pivoting away from the predator once it has committed to its attack. Remarkably, this antipredator strategy reverses the commonly observed negative scaling relationship between prey size and evasive ability, with larger fish benefiting most from diverting predators. Taken together, our results introduce a new antipredator divertive strategy that may be widely used by conspicuously colored prey that rely on agility to escape their predators., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Our purpose is to describe a safe and easy technique for the removal of the BrightOcular cosmetic iris implants. Our technique involves cutting the implant into five parts rather than removal as a whole with less intraocular manipulation and no rotation of the implant., Methods: We have used this technique in two eyes of a 28-year-old patient who presented to us with progressive drop of vision and acute attacks of ocular pain. Best-corrected distance visual acuity (BCVA) was found to be 6/18 and 6/9 on Snellen chart in her right and left eyes, respectively. Intraocular pressure was 40 mmHg and 20 mmHg in the right and left eyes, respectively, with visual field and retinal nerve fiber layer changes in the right eye., Results: After removal of both implants, 1-week post-operative BCVA improved to 6/6 bilaterally. After 3 months of regular follow-ups, IOP remains uncontrolled in the right eye despite medical treatment, and surgical intervention is planned., Conclusion: Our five-slice technique for removal of iris implants can be done effectively through a small corneal wound thus avoiding most wound-related problems. Our technique also includes no rotation or excessive manipulation inside the anterior chamber and can be carried out easily and safely with both the NewColorIris and BrightOcular implant designs.

Objective: A genome-wide high-throughput single nucleotide polymorphism (SNP) typing method was tested with respect of the applicability to ancient and degraded DNA. The results were compared to mini-sequencing data achieved through single base extension (SBE) typing. The SNPs chosen for the study allow to determine the hair colors and eye colors of humans., Material and Methods: The DNA samples were extracted from the skeletal remains of 59 human individuals dating back to the Late Bronze Age. The 3,000 years old bones had been discovered in the Lichtenstein Cave in Lower Saxony, Germany. The simultaneous typing of 24 SNPs for each of the ancient DNA samples was carried out using the 192.24 Dynamic Array™ by Fluidigm®., Results: Thirty-eight of the ancient samples (=64%) revealed full and reproducible SNP genotypes allowing hair and eye color phenotyping. In 10 samples (=17%) at least half of the SNPs were unambiguously determined, in 11 samples (=19%) the SNP typing failed. For 23 of the 59 individuals, a comparison of the SNP typing results with genotypes from an earlier performed SBE typing approach was possible. The comparison confirmed the full concordance of the results for 90% of the SNP typings. In the remaining 10% allelic dropouts were identified., Discussion: The high genotyping success rate could be achieved by introducing modifications to the preamplification protocol mainly by increasing the DNA input and the amplification cycle number. The occurrence of allelic dropouts indicates that a further increase of DNA input to the preamplification step is desirable., (© 2020 The Authors. American Journal of Physical Anthropology published by Wiley Periodicals, Inc.)

Background: In an effort to alter eye color during World War II, devout Nazi researcher Karin Magnussen had adrenaline eye drops administered to inmates at the concentration camp Auschwitz-Birkenau. A Sinti family, with a high prevalence of heterochromia iridis, was forced to participate in this study. Members of this family, as well as other victims, were later killed and had their eyes enucleated and sent to Magnussen for examination. Magnussen articulated the findings of these events in a manuscript that has never been published. The author is the first ophthalmologist to review this manuscript. The generation who experienced the atrocities of World War II will soon be gone and awareness of what happened during this tragic chapter of world history is fading., Objectives: To describe these events to raise awareness among future generations., Methods: A literature review and archival search was conducted., Results: Magnussen's research was based on an animal study published in 1937. For Magnussen's study, adrenaline drops were administered to inmates, including a 12-year-old girl from the Sinti family. As there was a reported case of deaf-mutism within the family, Waardenburg syndrome seems to be the most plausible explanation for this family's heritable heterochromia., Conclusions: The effort to change eye color was doomed to fail from the beginning because there was a probable diagnosis of Waardenburg syndrome. Extinction of humans for ophthalmological research is an insane act beyond imagination. For the sake of these victims, and for the generations who still feel their pain, it is imperative to tell their stories.

Research on the perception of faces typically assumes that there are some universal values of attractiveness which are shared across individuals and cultures. The perception of attractiveness may, however, vary across cultures due to local differences in both facial morphology and standards of beauty. To examine cross-cultural consensus in the ratings of attractiveness, we presented a set of 120 non-manipulated photographs of Czech faces to ten samples of raters from both European (Czech Republic, Estonia, Sweden, Romania, Turkey, Portugal) and non-European countries (Brazil, India, Cameroon, Namibia). We examined the relative contribution of three facial markers (sexual shape dimorphism, averageness, fluctuating asymmetry) to the perception of attractiveness as well as the possible influence of eye color, which is a locally specific trait. In general, we found that both male and female faces which were closer to the average and more feminine in shape were regarded as more attractive, while fluctuating asymmetry had no effect. Despite a high cross-cultural consensus on attractiveness standards, significant differences in the perception of attractiveness seem to be related to the level of socio-economic development (as measured by the Human Development Index, HDI). Attractiveness ratings by raters from low-HDI countries (India, Cameroon, Namibia) converged less with ratings from Czech Republic than ratings from high-HDI countries (European countries and Brazil). With respect to eye color, some local patterns emerged which we discuss as a consequence of negative frequency-dependent selection., Competing Interests: The authors have declared that no competing interests exist.

Purpose: To report outcomes of keratopigmentation (KP) with commercial black ink in the treatment of dysphotopsia secondary to laser peripheral iridotomies (LPI) using manual anterior stromal puncture (ASP) and manual lamellar pocket (LP)., Methods: This is a retrospective case series of eyes that underwent KP for treatment of dysphotopsia secondary to LPI. Patients' postoperative symptoms were categorized as resolved, improved, no change, or worse. Any intraoperative and postoperative complications were noted, as well as the need for further treatments., Results: Five eyes in 4 patients underwent ASP, and 14 eyes in 13 patients underwent LP. Only 1 patient had improvement in symptoms in the AK group. Four patients had a complete resolution of symptoms after LP, whereas 7 had symptomatic improvement and 3 did not notice any change. Fifty-five percent of patients in the LP group experienced late-onset depigmentation between 3 and 5 years postoperatively. One patient who underwent 2 LP re-treatments experienced irregular corneal steepening with nonprogressive corneal thinning. Overall, there were no serious adverse reactions to the pigment used., Conclusions: ASP was not suitable for the treatment of dysphotopsia secondary to LPI. In the short term, LP had good outcomes, but commercial black ink was prone to depigmentation at 3 to 5 years postoperatively. We therefore do not recommend the use of such pigment for long-term management of dysphotopsia secondary to LPI.

Forensic DNA Phenotyping (FDP) provides the ability to predict externally visible characteristics from minute amounts of crime scene DNA, which can help find unknown perpetrators who are typically unidentifiable via conventional forensic DNA profiling. Fundamental human genetics research has led to a better understanding of the specific DNA variants responsible for physical appearance characteristics, particularly eye, hair, and skin color. Recently, we introduced the HIrisPlex-S system for the simultaneous prediction of eye, hair, and skin color based on 41 DNA variants generated from two forensically validated SNaPshot multiplex assays using capillary electrophoresis (CE). Here we introduce massively parallel sequencing (MPS) solutions for the HIrisPlex-S (HPS) system on two MPS platforms commonly used in forensics, Ion Torrent and MiSeq, that cover all 41 DNA variants in a single assay, respectively. Additionally, we present the forensic developmental validation of the two HPS-MPS assays. The Ion Torrent MPS assay, based on Ion AmpliSeq technology, illustrated the successful generation of full HIrisPlex-S genotypic profiles from 100 pg of input control DNA, while the MiSeq MPS assay based on an in-house design yielded complete profiles from 250 pg of input DNA. Assessing simulated forensic casework samples such as saliva, hair (bulb), blood, semen, and low quantity touch DNA, as well as artificially damaged DNA samples, concordance testing, and samples from numerous species, all illustrated the ability of both versions of the HIrisPlex-S MPS assay to produce results that motivate forensic applications. By also providing an integrated bioinformatics analysis pipeline, MPS data can now be analyzed and a file generated for upload to the publically accessible HIrisPlex online webtool (https://hirisplex.erasmusmc.nl). In addition, we updated the website to accept VCF input data for those with genome sequence data. We thus provide a user-friendly and semi-automated MPS workflow from DNA sample to individual eye, hair, and skin color prediction probabilities. Furthermore, we present a 2-person mixture separation tool that not only assesses genotype reliability with regards genotyping confidence but also provides the most fitting mixture scenario for both minor and major contributors, including profile separation. We envision this MPS implementation of the HIrisPlex-S system for eye, hair, and skin color prediction from DNA as a starting point for further expanding MPS-based forensic DNA phenotyping. This may include the future addition of SNPs predictive for more externally visible characteristics, as well as SNPs for bio-geographic ancestry inference, provided the statistical framework for DNA prediction of these traits is in place., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose : To evaluate the refractive outcome and influencing factors following atropine 0.5% eye-drops applied twice daily during 2 ½ days at home and two drops of cyclopentolate 1% (C+C) and one drop of cyclopentolate 1% and one drop of tropicamide 1% (C+T) applied in an outpatient clinic, in hypermetropic children with a dark iris. Methods : Double-blind randomized study including 67 3-6-year-old children receiving C+C in one eye and C+T in the other eye. Two weeks later followed by atropine 0.5% in both eyes. Primary outcome measures were: spherical equivalent (SEQ) following C+C, C+T and atropine, and secondarily SEQ with respect to sex, ethnicity, skin pigmentation (light, medium, dark) and crying. Data on atropine are divided in those with C+C (CC) or C+T (CT) as a first intervention. Results : Mean SEQ±SD for C+C, C+T, atropine-(CC) and atropine-(CT) was +1.74 ± 1.35, +1.77 ± 1.34, +2.15 ± 1.43 and +2.10 ± 1.38 diopter (D). Atropine 0.5% revealed significantly more hypermetropia than C+C and C+T; +0.41 ± 0.43, 95%CI +0.31 to +0.52D and +0.33 ± 0.39, 95%CI +0.24 to +0.34D. No significant difference was present between C+C and C+T; -0.03 ± 0.56, 95%CI -0.16 to +0.11D. Ethnicity and skin-color were strongly associated (r = 0.84, p < .001). Sex was not affecting outcomes ( p = .101). Ethnicity was borderline significant ( p = .049). Skin-color was a highly significant factor ( p = .002). A statistical model combining intervention and skin-color, with light-pigmented subjects receiving atropine-(CC) as reference group (mean SEQ +2.61 ± 1.46D), indicated borderline significantly less hypermetropia in atropine-(CC)-dark: mean decrease (95%CI): -0.81 (-1.66 to +0.05)D and atropine-(CT)-dark -0.87 (-1.70 to -0.03)D, furthermore significantly less hypermetropia in C+C-dark: -1.15 (-1.97 to -0.32)D; C+T-dark: -1.21 (-2.03 to -0.39)D, C+C-medium: -1.02 (-1.81 to -0.24)D and C+T-medium: -0.86 (-1.64 to -0.08)D. Adding crying to the model significantly less hypermetropia was found for subjects crying in all interventions; -0.53 (-0.98 to -0.09)D. Within the interventions, with light-pigmented non-crying subjects as reference group (mean SEQ in atropine, C+C, respectively, C+T: +2.62 ± 1.41, +2.33 ± 1.20 and +2.32 ± 1.20D), showed significantly less hypermetropia in dark-pigmented crying subjects in each individual intervention: atropine -1.10 (-2.01 to -0.19), C+C -1.28 (-2.14 to -0.42) and C+T -1.34 (-2.20 to -0.48)D. For medium pigmented crying subjects this was present in atropine: -0.82 (-1.61 to -0.03)D and C+C: -0.86 (-1.68 to -0.04)D, but not in C+T: -0.58 (-1.25 to +0.09)D. Conclusions : Atropine 0.5% revealed a slight significantly higher hypermetropia. A dark-pigmented skin, especially when crying upon application, resulted in lower hypermetropia in all interventions. C+T provided clinically better results in medium pigmented crying subjects compared to C+C, and equal results compared to atropine 0.5%.

A 24-year-old Iranian woman presented to our department for routine eye control. She had had cosmetic iris implants (BrightOcular©) implanted 5 years before in a Tunisian eye clinic in order to change the color of her eyes. Despite subjective freedom of symptoms, a dramatically reduced corneal endothelial cell count was measured in the left eye. Therefore, surgical removal of the angle-supported cosmetic iris implants was necessary. Cosmetic iris implants are associated with sight-threatening complications-even years after their implantation. We report on a case of cosmetic iris implants inserted for alteration of eye color only, which gave rise to serious complications for our patient.

Genetics and environmental factors frequently influence individual's susceptibility to hearing loss. It is postulated that melanin in the inner ear is related to individual's susceptibility to noise induced hearing loss (NIHL). General pigmentation in turn, suspected to be related to the amount of pigmentation in the inner ear. The amount of melanin in the inner ear is said to modulate the endocochlear potential and provide an otoprotective effect., Aim: The study aimed to determine the relationship between the contralateral suppression of otoacoustic emissions (CSOAE) in individuals with brown eyes and hair, and blue eyes and blond hair, and temporary emission shift (TES) after short-term noise exposure., Setting and Design: The research was conducted using a quantitative research design with a quasi-experimental repeated within the subject design to compare the CSOAE in subjects with different hair and eyes colour with TES after short-term noise exposure. Quantitative research was used to determine the relationship between the measurable variables to predict occurrence., Material and Method: The hearing sensitivity of young adults was determined by using pure tone audiometry followed by CSOAE's and distortion product otoacoustic emissions (DPOAE) before listening to music for one hour individually. Pure tone audiometry and DPOAE's were repeated after music exposure to determine the amount of TES and temporary threshold shift (TTS)., Statistical Analysis Used: One-way ANOVA was used during the analysis of the data obtained during this research study, in addition to, two-tailed Wilcoxon Sign Rank test and Friedman's test. In all analyses, a 95% level of significance (P<0.05) was used., Results: No statistically significant difference between efferent suppression was measured by CSOAE's between the participant groups. A larger TTS at 4000 Hz and TES at 2000 Hz was evident in the blue eyes and blond hair group after short-term music exposure. Conclusion: CSOAE's were unable to predict which group of individuals were more susceptible to NIHL after short-term noise exposure., Competing Interests: None

This study examined 266 individuals from various populations including African American, East Asian, South Asian, European, and mixed populations to evaluate the ForenSeq™ Signature Prep Kit Primer Mix B. Focus was placed on phenotypic and biogeographical ancestry predictions by Illumina's Universal Analysis Software (UAS). These outcomes were compared to those obtained through web-tools developed at the Erasmus Medical Center (EMC) and available from the Forensic Resource/Reference on Genetics-knowledge base (FROG-kb), as well as to eye color predictions by the 8-plex system. Due to drop-outs, predictions for eye and hair color by UAS failed for various samples in each run. By including reads below thresholds, predictions could be obtained for all samples through the web-tools. Eye and hair color predictions for African Americans, East Asians, and South Asians showed no errors. Difficulties however, were noted in intermediate (neither blue nor brown) eye color predictions. These were mitigated by the 8-plex system through exclusion of one eye color (e.g. "not brown"). Additionally, notable discrepancies were observed in hair color predictions, where some black/dark-brown haired individuals were predicted to have blond hair. Overall, ancestry predictions were more accurate by FROG-kb compared to UAS, which did not predict South Asian ancestry, particularly Indian individuals., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To explore the temporal pattern of postmortem color changes in the pupil region of the cornea for noninvasive estimation of the postmortem interval (PMI)., Methods: Two rabbit models of air embolism and drowning were established in a dark room at a temperature of 20 ℃ with a relative humidity of 30%. The corneal images of the rabbits were acquired using a digital camera at two-hour intervals within 72 h after death. The pupil region on the corneal images was segmented using computer image processing technique (MATLAB), and the parameters of 6 image color features (RGBHSV) were extracted. Regression analysis was used to analyze the relationship between these parameters and the PMI, and the effects of different death causes on the changes of the corneal color features were also assessed., Results: Within 72 h after death from different causes, the R, G and B values of the pupil region on the corneal images all tended to increase with the PMI, showing a good fitting with the PMI ( P &lt; 0.01). No significant correlation was found between the values of H, S and V and the PMI ( P &gt;0.05). The R, G and B values in the pupil region on the corneal images showed consistent variation trends after death from the two causes, and their correlations with PMI were also similar. The measured values of R, G and B in air embolism group were greater than those in the drowning group., Conclusions: The postmortem color changes of the pupil region on corneal images follow an identifiable temporal pattern and can vary across different causes of death. The regression equations established in this study provide references for non-invasive and objective estimation of the PMI.

Fish can change their skin and eye colour for background matching and signalling. Males of Gasterosteus aculeatus develop ornamental blue eyes and a red jaw during the reproductive season, colours that are further enhanced during courtship. Here, the effects of different hormones on physiological colour changes in the eyes and jaws of male and female G. aculeatus were investigated in vitro. In an in vivo experiment, G. aculeatus were injected with a receptor blocker of a pivotal hormone (noradrenaline) that controls colour change. In males, noradrenaline had aggregating effects on melanophore and erythrophore pigments resulting in blue eyes and a pale jaw, whereas melanocyte-concentrating hormone (MCH) and melatonin resulted in a pale jaw only. When noradrenalin was combined with melanocyte stimulating hormone (MSH) or prolactin, the jaw became red, while the eyes remained blue. In vivo injection of yohimbine, an alpha-2 adrenoreceptor blocker, resulted in dispersion of melanophore pigment in the eyes and inhibited the blue colouration. Altogether, the data suggest that noradrenalin has a pivotal role in the short-term enhancement of the ornamental colouration of male G. aculeatus, potentially together with MSH or prolactin. This study also found a sex difference in the response to MCH, prolactin and melatonin, which may result from different appearance strategies in males, versus the more cryptic females., (© 2018 The Fisheries Society of the British Isles.)

Introduction: Recent studies have investigated the relationship between pain perception and specific phenotypes such as red hair color and various eye colors. Further investigations into biomarkers as they relate to pain could be useful in understanding underlying genetic components involved in these pathways. Additionally, it would be clinically useful to determine if a patient would be more likely to experience pain during dental treatment based on eye color. The purpose of this study was to investigate a link between eye color and perceived injection pain in healthy, asymptomatic white women., Methods: Three hundred healthy, adult, white female patients were included, 133 with dark eyes and 167 with light eyes. Dental anxiety was assessed with the Corah Dental Anxiety Scale. Subjects with their eye color masked by dark glasses received a right maxillary lateral incisor infiltration of 1 cartridge of 2% lidocaine with 1:100,000 epinephrine. Patients rated their injection pain on a 170-mm Heft-Parker visual analog scale. Photographs of the subjects' eyes were taken after the infiltrations and categorized into dark- and light-eyed groups by 3 independent observers. Comparisons for injection pain were analyzed using analysis of variance and the Tukey-Kramer test., Results: No significant differences were found for pain of injection between dark- or light-eyed subjects., Conclusions: Eye color was not shown to be a predictor for injection pain in white women. Therefore, eye color would not be clinically useful in determining if a patient would be more likely to experience pain during dental treatment., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

For historic individuals, the outward appearance and other phenotypic characteristics remain often non-resolved. Unfortunately, images or detailed written sources are only scarcely available in many cases. Attempts to study historic individuals with genetic data so far focused on hypervariable regions of mitochondrial DNA and to some extent on complete mitochondrial genomes. To elucidate the potential of in-solution based genome-wide SNP capture methods - as now widely applied in population genetics - we extracted DNA from the 17th century remains of George Bähr, the architect of the Dresdner Frauenkirche. We were able to identify the remains to be of male origin, showing sufficient DNA damage, deriving from a single person and being thus likely authentic. Furthermore, we were able to show that George Bähr had light skin pigmentation and most likely brown eyes. His genomic DNA furthermore points to a Central European origin. We see this analysis as an example to demonstrate the prospects that new in-solution SNP capture methods can provide for historic cases of forensic interest, using methods well established in ancient DNA (aDNA) research and population genetics.

Purpose: Understanding the association of iris colour and myopia may provide further insights into the role of the wavelength of lights in the pathophysiology of myopia. We aim to assess the association of iris colour and myopia in a school-based sample of Chinese students., Methods: Two thousand three hundred and forty-six Year 7 students from 10 middle schools (93.5% response rate) aged 13-14 years in Mojiang, a small county located in Southwestern China, participated in the study. We obtained standardised slit lamp photographs and developed a grading system assessing iris colour (higher grade denoting a darker iris). Refractive error was measured after cycloplegia using an autorefractor by optometrists or trained technicians. An IOLMaster (www.zeiss.com) was used to measure ocular biometric parameters including axial length (AL)., Results: Of all the study participants, 693 (29.5%) were affected by myopia with the prevalence estimates being higher in girls (36.8%; 95% confidence interval [CI]: 34.0, 39.6) than in boys (22.8%; 95% CI: 20.4, 25.1) (p < 0.001). After adjusting for gender, height, parental history of myopia, time spent on computer, time spent watching TV, time spent outdoors, and time spent reading and writing, participants with a darker iris colour tended to have a higher prevalence of myopia, a more myopic refraction and a longer AL. Dose-response relationships were observed in all regression models (p for trend <0.05)., Conclusions: Darker iris colour was associated with more myopic refractive errors and longer ALs among Chinese school-aged children and this association was independent of other known myopia-related risk factors., (© 2017 The Authors Ophthalmic & Physiological Optics © 2017 The College of Optometrists.)

Retinaldehyde adducts (bisretinoids) accumulate in retinal pigment epithelial (RPE) cells as lipofuscin. Bisretinoids are implicated in some inherited and age-related forms of macular degeneration that lead to the death of RPE cells and diminished vision. By comparing albino and black-eyed mice and by rearing mice in darkness and in cyclic light, evidence indicates that bisretinoid fluorophores undergo photodegradation in the eye (Ueda et al. Proc Natl Acad Sci 113:6904-6909, 2016). Given that the photodegradation products modify and impair cellular and extracellular molecules, these processes likely impart cumulative damage to retina.

Red hair is associated in women with pain sensitivity. This medical condition, and perhaps others, seems facilitated by the combination of being red-haired and female. We tested this hypothesis by questioning a large sample of Czech and Slovak respondents about the natural redness and darkness of their hair, their natural eye color, their physical and mental health (24 categories), and other personal attributes (height, weight, number of children, lifelong number of sexual partners, frequency of smoking). Red-haired women did worse than other women in ten health categories and better in only three, being particularly prone to colorectal, cervical, uterine, and ovarian cancer. Red-haired men showed a balanced pattern, doing better than other men in three health categories and worse in three. Number of children was the only category where both male and female redheads did better than other respondents. We also confirmed earlier findings that red hair is naturally more frequent in women than in men. Of the 'new' hair and eye colors, red hair diverges the most from the ancestral state of black hair and brown eyes, being the most sexually dimorphic variant not only in population frequency but also in health status. This divergent health status may have one or more causes: direct effects of red hair pigments (pheomelanins) or their by-products; effects of other genes that show linkage with genes involved in pheomelanin production; excessive prenatal exposure to estrogen (which facilitates expression of red hair during fetal development and which, at high levels, may cause health problems later in life); evolutionary recentness of red hair and corresponding lack of time to correct negative side effects; or genetic incompatibilities associated with the allele Val92Met, which seems to be of Neanderthal origin and is one of the alleles that can cause red hair.