Evolution repeats itself in replicate long-term studies in the wild.

The extent to which evolution is repeatable remains debated. Here, we study changes over time in the frequency of cryptic color-pattern morphs in 10 replicate long-term field studies of a stick insect, each spanning at least a decade (across 30 years of total data). We find predictable "up-and-down" fluctuations in stripe frequency in all populations, representing repeatable evolutionary dynamics based on standing genetic variation. A field experiment demonstrates that these fluctuations involve negative frequency-dependent natural selection (NFDS). These fluctuations rely on demographic and selective variability that pushes populations away from equilibrium, such that they can reliably move back toward it via NFDS. Last, we show that the origin of new cryptic forms is associated with multiple structural genomic variants such that which mutations arise affects evolution at larger temporal scales. Thus, evolution from existing variation is predictable and repeatable, but mutation adds complexity even for traits evolving deterministically under natural selection.

DNA methylation differences between stick insect ecotypes.

Epigenetic mechanisms, such as DNA methylation, can influence gene regulation and affect phenotypic variation, raising the possibility that they contribute to ecological adaptation. Beginning to address this issue requires high-resolution sequencing studies of natural populations to pinpoint epigenetic regions of potential ecological and evolutionary significance. However, such studies are still relatively uncommon, especially in insects, and are mainly restricted to a few model organisms. Here, we characterize patterns of DNA methylation for natural populations of Timema cristinae adapted to two host plant species (i.e. ecotypes). By integrating results from sequencing of whole transcriptomes, genomes and methylomes, we investigate whether environmental, host and genetic differences of these stick insects are associated with methylation levels of cytosine nucleotides in the CpG context. We report an overall genome-wide methylation level for T. cristinae of ~14%, with methylation being enriched in gene bodies and impoverished in repetitive elements. Genome-wide DNA methylation variation was strongly positively correlated with genetic distance (relatedness), but also exhibited significant host-plant effects. Using methylome-environment association analysis, we pinpointed specific genomic regions that are differentially methylated between ecotypes, with these regions being enriched for genes with functions in membrane processes. The observed association between methylation variation and genetic relatedness, and with the ecologically important variable of host plant, suggests a potential role for epigenetic modification in T. cristinae adaptation. To substantiate such adaptive significance, future studies could test whether methylation can be transmitted across generations and the extent to which it responds to experimental manipulation in field and laboratory studies.

Complex evolutionary processes maintain an ancient chromosomal inversion.

Genome re-arrangements such as chromosomal inversions are often involved in adaptation. As such, they experience natural selection, which can erode genetic variation. Thus, whether and how inversions can remain polymorphic for extended periods of time remains debated. Here we combine genomics, experiments, and evolutionary modeling to elucidate the processes maintaining an inversion polymorphism associated with the use of a challenging host plant (Redwood trees) in Timema stick insects. We show that the inversion is maintained by a combination of processes, finding roles for life-history trade-offs, heterozygote advantage, local adaptation to different hosts, and gene flow. We use models to show how such multi-layered regimes of balancing selection and gene flow provide resilience to help buffer populations against the loss of genetic variation, maintaining the potential for future evolution. We further show that the inversion polymorphism has persisted for millions of years and is not a result of recent introgression. We thus find that rather than being a nuisance, the complex interplay of evolutionary processes provides a mechanism for the long-term maintenance of genetic variation.

Epigenetic effects of climate change on insects.

Climate change has been causing severe modifications to the environment that are predicted to aggravate in the future, which create critical challenges for insects to cope. Populations can respond to the changes depending on the standing genetic variation. Additionally, they could potentially rely on epigenetic mechanisms as a source of phenotypic variation. These mechanisms can influence gene regulation and can respond to the external environment, being implicated in phenotypic plasticity. Thus, epigenetic variation could be advantageous in changing, unpredictable environments. However, little is known about causal relationships between epigenetic marks and insects' phenotypes, and whether the effects are truly beneficial to the fitness. Empirical studies are now urgent to better understand whether epigenetic variation can help or hinder insect populations facing climate change.

Disruptive selection and the evolution of discrete color morphs in Timema stick insects.

A major unresolved issue in biology is why phenotypic and genetic variation is sometimes continuous, yet other times packaged into discrete units of diversity, such as morphs, ecotypes, and species. In theory, ecological discontinuities can impose strong disruptive selection that promotes the evolution of discrete forms, but direct tests of this hypothesis are lacking. Here, we show that Timema stick insects exhibit genetically determined color morphs that range from weakly to strongly discontinuous. Color data from nature and a manipulative field experiment demonstrate that greater morph differentiation is associated with shifts from host plants exhibiting more continuous color variation to those exhibiting greater coloration distance between green leaves and brown stems, the latter of which generates strong disruptive selection. Our results show how ecological factors can promote discrete variation, and we further present results on how this can have variable effects on the genetic differentiation that promotes speciation.

A crossover study assessing the protective efficacy of improvised snorkel-based improvised respirators.

Introduction: This study was designed to determine whether improvised respirators based on modified full-face snorkel masks are able to pass a standard qualitative fit test. Methods: This is a prospective crossover study conducted in 16 staff. Fit-tests were conducted on masks mated to (1) an anaesthetic breathing circuit heat and moisture exchange filter and (2) a CE-marked P3 grade filter. P3 filters were mounted using both epoxy-coated and uncoated adaptors. Results: None of the tests using anaesthetic filters passed. Only one overall pass was observed using the P3-rated filter mated to the snorkel mask. Conclusions: These data suggest that improvised PPE designs cannot provide reliable protection against aerosols. Failures are likely due to poor fit, but the suitability of 3D printed materials is also uncertain as fused-filament manufacturing yields parts that are not reliably gas-tight. Improvised PPE cannot be recommended as a substitute for purpose designed systems.

Testing for fitness epistasis in a transplant experiment identifies a candidate adaptive locus in Timema stick insects.

Identifying the genetic basis of adaptation is a central goal of evolutionary biology. However, identifying genes and mutations affecting fitness remains challenging because a large number of traits and variants can influence fitness. Selected phenotypes can also be difficult to know a priori, complicating top-down genetic approaches for trait mapping that involve crosses or genome-wide association studies. In such cases, experimental genetic approaches, where one maps fitness directly and attempts to infer the traits involved afterwards, can be valuable. Here, we re-analyse data from a transplant experiment involving Timema stick insects, where five physically clustered single-nucleotide polymorphisms associated with cryptic body coloration were shown to interact to affect survival. Our analysis covers a larger genomic region than past work and revealed a locus previously not identified as associated with survival. This locus resides near a gene, Punch (Pu), involved in pteridine pigments production, implying that it could be associated with an unmeasured coloration trait. However, by combining previous and newly obtained phenotypic data, we show that this trait is not eye or body coloration. We discuss the implications of our results for the discovery of traits, genes and mutations associated with fitness in other systems, as well as for supergene evolution. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

A crossover trial investigating the atmospheric content of improvised respirators.

Introduction: This study was designed to determine the respiratory safety of improvised respirators based on modified full-face snorkel masks, making comparisons with a purpose-designed mask. Methods: This is a prospective crossover study conducted on ten recruits. Volunteers wore snorkel masks mated to an anaesthetic heat and moisture exchange filter. The system was worn at rest then during exercise. Gases were sampled from the mask at 5-min intervals. Results: The modified snorkel was satisfactory in seven participants. For three carbon dioxide concentrations were >1%. Two participants exposed to excessive CO2 also experienced oxygen concentrations <19%. All participants exposed to unsatisfactory gas mixtures were non-white. Conclusions: Modifying snorkel masks changes the way that gases circulate through the system. These modifications increase the risk of rebreathing in some users, which may yield an unsafe gas mixture. These improvised masks cannot be recommended as a substitute for purpose-designed equipment.

Laparoscopy as a Facilitator in the Early Diagnosis of a Mullerian Aplasia During Ordinary Surgery.

Laparoscopy was introduced more than 100 years ago. However, in some fields its use still meets resistance. Technology such as laparoscopy may help to identify rare and complex disorders, even in very ordinary procedures, such as inguinal hernia repair. This report highlighted the importance of early diagnosis of a complex condition using commonly available technology. To the best of our knowledge, there has not been a similar reported case in such a young patient during laparoscopic inguinal hernia repair.

The eye of the giant anteater (Myrmecophaga tridactyla): Ophthalmic examination findings and diagnostic tests.

OBJECTIVE: To describe ophthalmic examination and diagnostic values for Schirmer tear test (STT), intraocular pressure (IOP), corneal horizontal diameter (CHD), palpebral fissure length (PFL), fundoscopy, and palpebral conjunctiva microbiota from healthy giant anteaters. ANIMALS STUDIED: Twelve giant anteaters (Myrmecophaga tridactyla), 11 adults and one juvenile, five males and seven females. PROCEDURES: The animals were submitted to general anesthesia and ophthalmic evaluation with portable slit-lamp biomicroscope, Finoff transilluminator, and fundoscopy, as well as STT, bacterial culture from palpebral conjunctiva, rebound tonometry IOP, and measurement of PFL and CHD. Data compiled were analyzed with ANOVA and Tukey tests. RESULTS: The results (mean ± standard deviation) were as follows: STT 8.04 ± 6.21 mm/min; IOP 10.92 ± 2.45 mmHg; PFL 0.75 ± 0.11 cm; CHD 0.96 ± 0.10 cm. Out of the 24 eyes swab samples for bacterial culture, 17 were positive, with three genera of Gram-positive bacteria identified Staphylococcus spp., Bacillus sp., and Corynebacterium sp. Gram-negative bacteria were not isolated from any of the samples. CONCLUSIONS: As conservation work in this vulnerable species continues, this report on basic ophthalmic examination and diagnostic parameters will be helpful improve their treatment and care. More ophthalmic studies are encouraged in animals within the Pilosa order.

Spontaneous Pubertal Onset in a Male Patient With Mixed Gonadal Dysgenesis With Mosaicism 45,X/ 46, X, mar (Y)/ 47,X,mar(Y),+mar(Y) - Pediatric Case Report.

This report describes an adolescent with Mixed Gonadal Dysgenesis and unexpected mosaicism [karyotype 46,X,mar(Y)/ 47,X, mar(Y),+mar(Y)].). Diagnosis with 1 month of age due to atypical genitalia. He presented a right streak gonad, which was removed due to the risk for germ cell tumor, and a left testis with epididymis barely connected and without vas deferens. Left testis maintenance was sufficient for him to undergo spontaneous puberty. The patient was non-responsive to growth hormone. Webbed neck was the only dysmorphic feature. To the best of our knowledge, there were no similar cases reported with spontaneous pubertal progress reported in the literature.

Laparoscopic gonad-sparing procedure in Ovotesticular disorder of sex development - Case video technique.

INTRODUCTION: Ovotesticular disorder of sex development (OTD) is a rare condition. There's a lack of literature addressing gonad-sparing surgery for OTD. OBJECTIVE: Report the laparoscopic partial gonadectomy technique - gonad-sparing surgery - in an 11-year-old child, 46, XX karyotype with OTD with atypical genitalia. MATERIAL AND METHODS: After a complete diagnostic evaluation the patient underwent feminizing genitoplasty followed by laparoscopic partial gonadectomy (gonad-sparing surgery). The patient was positioned on supine position and Trendelenburg. One 5 mm port was placed on the umbilicus and two 3 mm ports in both flanks. A gonadal wedge biopsy was performed to achieve histopathological confirmation before resection. The testicular component of the ovotestis is clearly identified based on macroscopic aspects, and resected with laparoscopic scissors and limited use of electrocautery. DISCUSSION: This case was classified as bipolar or terminal ovotestis. At the 5-month follow-up, the patient attained menarche. No adverse outcomes have been recorded. Postoperative third year follow-up hormone evaluation revealed a= female pattern characteristic and ultrasound demonstraed uterine volume increase, as well as bilateral ovarian tissue development with follicles. CONCLUSIONS: Gonad-sparing procedure is feasible and desirable whenever possible, especially in 46, XX patients with female sex of rearing, since it preserves the fertility potential. The risk of malignancy must be monitored.

Prospective observational study of gender and ethnicity biases in respiratory protective equipment for healthcare workers in the COVID-19 pandemic.

OBJECTIVE: To describe success rates of respiratory protective equipment (RPE) fit testing and factors associated with achieving suitable fit. DESIGN: Prospective observational study of RPE fit testing according to health and safety, and occupational health requirements. SETTING: A large tertiary referral UK healthcare facility. POPULATION: 1443 healthcare workers undergoing quantitative fit testing. MAIN OUTCOME MEASURES: Quantitative fit test success (pass/fail) and the count of tests each participant required before successful fit. RESULTS: Healthcare workers were fit tested a median (IQR) 2 (1-3) times before successful fit was obtained. Males were tested a median 1 (1-2) times, while females were tested a median 2 (1-2) times before a successful fit was found. This difference was statistically significant (p<0.001). Modelling each fit test as its own independent trial (n=2359) using multivariable logistic regression, male healthcare workers were significantly more likely to find a well-fitting respirator and achieve a successful fit on first attempt in comparison to females, after adjusting for other factors (adjusted OR=2.07, 95% CI): 1.66 to 2.60, p<0.001). Staff who described their ethnicity as White were also more likely to achieve a successful fit compared with staff who described their ethnicity as Asian (OR=0.47, 95% CI: 0.38 to 0.58, p<0.001), Black (OR=0.54, 95% CI: 0.41 to 0.71, p<0.001), mixed (OR=0.50 95% CI: 0.31 to 0.80, p=0.004) or other (OR=0.53, 95% CI: 0.29 to 0.99, p=0.043). CONCLUSIONS: Male and White ethnicity healthcare workers are more likely to achieve RPE fit test success. This has broad operational implications to healthcare services with a large female and Black, Asian and minority ethnic group population. Fit testing is imperative in ensuring RPE effectiveness in protecting healthcare workers during the COVID-19 pandemic and beyond.

Ocular examination findings and measurements of tear production and tonometry of ring-tailed coatis (nasua nasua).

OBJECTIVE: To describe ocular anatomical features and to determine tear production and tonometry in normal anesthetized ring-tailed coati (Nasua nasua). ANIMALS: Eight male and seven female ring-tailed coatis (30 eyes) were evaluated, with ages ranging from 7 months to 10 years old. PROCEDURES: The animals were anesthetized with isoflurane and had STT 1, rebound and applanation tonometry, and palpebral fissure length (PFL) measured in both eyes. Rebound tonometry was measured with both TonoVet® and TonoVet Plus®. All eyes were evaluated with slit-lamp biomicroscopy. Following pharmacological mydriasis, direct fundoscopy was performed. ANOVA and Tukey tests were used for the data analysis. RESULTS: There were upper and lower eyelids, as well as a third eyelid. Eyelashes were present on the upper eyelid, while meibomian glands openings were visible on both. The bulbar conjunctiva was pigmented. The pupil was obliquely horizontally ovoid and teardrop shaped when miotic. The color of the iris and tapetum lucidum varied with age. The optic disk was round, and the retina was holangiotic. The mean ± standard deviation values obtained were as follows: STT: 2.50 ± 2.39 mm/min; rebound tonometry: 9.50 ± 2.43 mmHg (TonoVet®, calibration P), 14.60 ± 2.82 mmHg (TonoVet®, calibration D), 18.93 ± 3.99 mmHg (TonoVet Plus®, calibration dog); applanation tonometry: 18.66 ± 4.16 mmHg; PFL: 10.98 ± 1.67 mm. CONCLUSIONS AND CLINICAL RELEVANCE: Anatomical and clinical ophthalmic data presented in this study are unprecedented for this species. The results can be used as clinical reference parameters, allowing for better care of these species. The lack of data for the Procyonidae family must be regarded as an incentive for more studies.

Influence of respiratory protective equipment on simulated advanced airway skills by specialist tracheal intubation teams during the COVID-19 pandemic.

Background: The COVID-19 pandemic has highlighted the importance of respiratory protective equipment for clinicians performing airway management. Aim: To evaluate the impact of powered air-purifying respirators, full-face air-purifying respirators and filtering facepieces on specially trained anaesthesiologists performing difficult airway procedures. Methods: All our COVID-19 intubation team members carried out various difficult intubation drills: unprotected, wearing a full-face respirator, a filtering facepiece or a powered respirator. Airway management times and wearer comfort were evaluated and analysed. Results: Total mean (SD) intubation times did not show significant differences between the control, the powered, the full-face respirator and the filtering facepiece groups: Airtraq 6.1 (4.4) vs. 5.4 (3.1) vs. 6.1 (5.6) vs. 7.7 (7.6) s; videolaryngoscopy 11.4 (9.0) vs. 7.7 (4.3) vs. 9.8 (8.4) vs. 12.7 (9.8) s; fibreoptic intubation 16.6 (7.8) vs.13.8 (6.7) vs. 13.6 (8.1) vs. 16.9 (9.2) s; and standard endotracheal intubation by direct laryngoscopy 8.1 (3.5) vs. 6.5 (5.6) vs. 6.2 (4.2) vs. 8.0 (4.4) s, respectively. Use of the Airtraq achieved the shortest intubation times. Anaesthesiologists rated temperature and vision significantly better in the powered respirator group. Conclusions: Advanced airway management remains unaffected by the respiratory protective equipment used if performed by a specially trained, designated team. We conclude that when advanced airway skills are performed by a designated, specially trained team, airway management times remain unaffected by the respiratory protective equipment used.

Ecology shapes epistasis in a genotype-phenotype-fitness map for stick insect colour.

Genetic interactions such as epistasis are widespread in nature and can shape evolutionary dynamics. Epistasis occurs due to nonlinearity in biological systems, which can arise via cellular processes that convert genotype to phenotype and via selective processes that connect phenotype to fitness. Few studies in nature have connected genotype to phenotype to fitness for multiple potentially interacting genetic variants. Thus, the causes of epistasis in the wild remain poorly understood. Here, we show that epistasis for fitness is an emergent and predictable property of nonlinear selective processes. We do so by measuring the genetic basis of cryptic colouration and survival in a field experiment with stick insects. We find that colouration shows a largely additive genetic basis but with some effects of epistasis that enhance differentiation between colour morphs. In terms of fitness, different combinations of loci affecting colouration confer high survival in one host-plant treatment. Specifically, nonlinear correlational selection for specific combinations of colour traits in this treatment drives the emergence of pairwise and higher-order epistasis for fitness at loci underlying colour. In turn, this results in a rugged fitness landscape for genotypes. In contrast, fitness epistasis was dampened in another treatment, where selection was weaker. Patterns of epistasis that are shaped by ecologically based selection could be common and central to understanding fitness landscapes, the dynamics of evolution and potentially other complex systems.

Large-scale mutation in the evolution of a gene complex for cryptic coloration.

The types of mutations affecting adaptation in the wild are only beginning to be understood. In particular, whether structural changes shape adaptation by suppressing recombination or by creating new mutations is unresolved. Here, we show that multiple linked but recombining loci underlie cryptic color morphs of Timema chumash stick insects. In a related species, these loci are found in a region of suppressed recombination, forming a supergene. However, in seven species of Timema, we found that a megabase-size "supermutation" has deleted color loci in green morphs. Moreover, we found that balancing selection likely contributes more to maintaining this mutation than does introgression. Our results show how suppressed recombination and large-scale mutation can help to package gene complexes into discrete units of diversity such as morphs, ecotypes, or species.

Ophthalmic contributions to assessing eyes of two neotropical canids: Cerdocyon thous and Chrysocyon brachyurus.

OBJECTIVE: To describe selected ophthalmic tests and anatomical features of eyes of crab-eating foxes (Cerdocyon thous) and maned wolves (Chrysocyon brachyurus). ANIMALS STUDIED: Six crab-eating foxes (12 eyes), eight maned wolves (16 eyes). PROCEDURES: Intramuscular and/or inhalatory anesthesia, ophthalmic evaluation with portable slit-lamp biomicroscope, Schirmer tear test (STT), intraocular pressure measurement with rebound and applanation (crab-eating fox only) tonometers, measurement of palpebral fissure length (PFL), gonioscopy, and fundoscopy. Data were analyzed with ANOVA and Tukey's tests. RESULTS: Both species presented upper and lower eyelids, both with eyelashes and Meibomian glands openings. A third eyelid was also present. In partial miosis, pigmented projections were observed along the edge of the pupil. The draining angle was open, with thin pectinate ligaments. The retina was holangiotic. For crab-eating foxes, mean ± standard deviation values were as follows: STT: 4.33 ± 2.96 mm/min; PFL: 17.45 ± 1.55 mm; rebound tonometry: 10.70 ± 3.43 mm Hg (TonoVet® calibration D),5.66 ± 3.44 mm Hg (TonoVet® calibration P), 17.00 ± 4.64 mm Hg (TonoVet® Plus calibration dog); and applanation tonometry: 11.70 ± 5.70 mm Hg(TonoPen® XL). For maned wolves, the mean ± standard deviation values were as follows: STT: 9.31 ± 7.40 mm/min; PFL: 22.79 ± 1.63 mm; rebound tonometry: 11.00 ± 2.77 mm Hg (TonoVet® calibration D), 6.78 ± 2.58 mm Hg (TonoVet® calibration P), and 18.29 ± 3.47 mm Hg(TonoVet® Plus calibration dog). CONCLUSIONS: This study contributes with knowledge that can help the clinical assessment regarding eyes of crab-eating foxes and maned wolves. The data herein presented for rebound tonometry are new for both species.