Where’s the Switch?

Sex change occurs as a usual part of the life cycle for many ray-finned fish, often following specific social cues. It has been shown that environmental factors can interact with, and sometimes override,genetic factors to control sexual development. More dramatically, in many marine fish, individuals can change sex as an adaptive response to environmental changes even during adulthood. Such sensitivity to environmental stimuli may explain why teleost or bony fishes display such highly diverse sex determination and developmental systems, which make them good models for understanding vertebrate sexual development. The exact mechanism behind the transduction of the environmental signals into the molecular cascade that underlies this singular transformation remains largely unknown. Cortisol is the main glucocorticoid in fish and the hormone most directly associated with stress. However, the exact role of cortisol or stress in transducing the external signals to elicit physiological responses during sexual development and sex change remains a mystery

Effects of cortisol on female-to-male sex change in a wrasse

Sex change occurs as a usual part of the life cycle for many teleost fish and the modifications involved (behavioural, gonadal, morphological) are well studied. However, the mechanism that transduces environmental cues into the molecular cascade that underlies this transformation remains unknown. Cortisol, the main stress hormone in fish, is hypothesised to be a key factor linking environmental stimuli with sex change by initiating gene expression changes that shift steroidogenesis from oestrogens to androgens but this notion remains to be rigorously tested. Therefore, this study aimed to experimentally test the role of cortisol as an initiator of sex change in a protogynous (female-to-male) hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). We also sought to identify potential key regulatory factors within the head kidney that may contribute to the initiation and progression of gonadal sex change. Cortisol pellets were implanted into female spotty wrasses under inhibitory conditions (presence of a male), and outside of the optimal season for natural sex change. Histological analysis of the gonads and sex hormone analyses found no evidence of sex change after 71 days of cortisol treatment. However, expression analyses of sex and stress-associated genes in gonad and head kidney suggested that cortisol administration did have a physiological effect. In the gonad, this included upregulation of amh, a potent masculinising factor, and nr3c1, a glucocorticoid receptor. In the head kidney, hsd11b2, which converts cortisol to inactive cortisone to maintain cortisol balance, was upregulated. Overall, our results suggest cortisol administration outside of the optimal sex change window is unable to initiate gonadal restructuring. However, our expression data imply key sex and stress genes are sensitive to cortisol. This includes genes expressed in both gonad and head kidney that have been previously implicated in early sex change in several sex-changing species

Impact of SARS-CoV-2 RNAemia and other risk factors on long-COVID: A prospective observational multicentre cohort study

As the COVID-19 pandemic has progressed, long-COVID has emerged as a major problem that poses a significant challenge for attending physicians and health care policy makers. Therefore, we read with much interest the recently published unicentre study in the Journal of Infection by Righi et al.,1 carried out on 465 adult COVID-19 patients (235 [50.5%] hospital-admitted) followed-up during nine months, concluding that those with advanced age, intensive care unit (ICU) admission, and multiple symptoms at onset were more likely to have long-term COVID-19 symptoms, with negative impact on physical and mental wellbeing. Other studies have found that female gender, age, longer hospital stay, pre-existing hypertension, cardiovascular disease, diabetes, chronic obstructive pulmonary disease, smoking, obesity, and chronic alcoholism increase the likelihood of long-COVID.2,3 It is known that SARS-CoV-2 RNAemia is a predictor of COVID-19 severity and in-hospital complications.4,5 However, to the best of our knowledge, only two studies have assessed, up to one or three months after the acute COVID-19 onset, whether SARS-CoV-2 RNAemia may have an impact on long-COVID,6,7 both finding that RNAemia at presentation might predict the persistence of symptoms. However, these studies did not provide information regarding long-COVID symptoms nor the association with SARS-CoV-2 RNAemia beyond three months, and could not differentiate between “true” long-COVID and the convalescence phase of the SARS-CoV-2 infection.A.R. has received a predoctoral research grant from the Instituto de Salud Carlos III, Spanish Ministry of Science, Innovation and Universities, (PFIS grant FI18/00183). G.A.A. reports a predoctoral research grant from the 201808-10 project, funded by La Marató de TV3. This study was supported by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, the Spanish Network for Research in Infectious Diseases (REIPI, RD16/0016/0001, RD16/0016/0005, RD16/0016/0009, RD16/0016/0013)-co-financed by the European Development Regional Fund, A way to achieve Europe, Operative program Intelligent Growth 2014-2020, and the Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC) [CB21/13/00009; CB21/13/00006], Madrid, Spain. J.S.C. and E.C. received grants from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Proyectos de Investigación sobre el SARS-CoV-2 y la enfermedad COVID-19 (COV20/00580; COV20/00370). J.S.C. is a researcher belonging to the program “Nicolás Monardes” (C-0059–2018), Servicio Andaluz de Salud, Junta de Andalucía, Spain. Samples and data from patients included in this study from the Hospital Universitario Cruces (Bizkaia, Spain) were provided by the Basque Biobank (www.biobancovasco.org) and were processed following standard operation procedures with appropriate approval of the Ethical and Scientific Committees.Peer reviewe

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Stress and sex change in New Zealand spotty wrasse (Notolabrus celidotus)

Sex is fixed from birth in most organisms, where individuals develop as either female or male and retain their sex through life. Traditionally, sex has been considered to be determined by genetic elements or by environmental factors. However, we now know numerous examples of plant and animal species that challenge this false dichotomy and where sexual fate is influenced by both strategies. One of the most fascinating examples of sexual plasticity is found in sequentially hermaphroditic fishes, in which species begin life as one sex, but can change to the other sometime thereafter. Considerable advances have been made in the understanding of the behavioural, morphological and molecular basis underlying this fascinating transformation. However, how cues received by fish (e.g. social cues, attainment of a certain size, etc.) trigger sex change at a physiological level is still not fully understood, and current research is biased towards tropical species. It has been hypothesised that this trigger is mediated by cross-talk between the hypothalamic-pituitary-gonadal (HPG) and -interrenal (HPI) axes, which regulate reproduction and stress, respectively. Cortisol has emerged as a potential key element in the transduction of environmental signals that can initiate sex change. A more comprehensive knowledge of this process is of considerable value, as not only will it shed light on our understanding of vertebrate sexual development, but may also be applied for sex control in aquaculture settings. This thesis aimed to characterise sex change in the temperate New Zealand spotty wrasse (Notolabrus celidotus), and highlight its potential to become a model for the study of sequential hermaphroditism. Spotties exhibit protogynous (female-to-male) sex change based on social context. Removal of the dominant male from a social group can induce sex change in the largest female. This thesis explored the physiological and genetic cascade underlying sex change in spotties, paying special attention to the role of cortisol, while also highlighting differences between tropical fishes that breed throughout the year and temperate fishes that breed seasonally. Experiments involving aromatase inhibition or social manipulation, within or outside the breeding season, were used to characterise the chemical and social induction of spotty sex change between 2014 – 2018. Alterations in gonadal morphology, steroid profile and gonadal candidate gene expression were explored. Measurement of plasma sex steroids 17β-estradiol (E2) and 11-ketotestosterone (11KT) across experiments showed a general trend of decreasing E2 and increasing 11KT towards maleness. A higher number of transitioning individuals were found among spotties manipulated outside their breeding season, when sex change occurs naturally. Timing of experiments relative to the breeding season also affected the steroid profile, and a drop in E2 concentrations at the onset of sex change, characteristic of tropical fish, was not observed in spotties during the post-spawning period, when sex steroids are expected to already be low. This suggested that arrested E2 production may not be a key event at the initiation of sex change in spotties, and potentially other temperate species. A panel of 21 genes were selected to explore the role of the sex determination and stress response pathways in the process of sex change using the nanoString platform. Principal Component Analysis of the patterns of gene expression revealed by our sex and stress gene panel in gonadal samples from fish socially induced to change sex revealed strong clustering of samples by sex-change stage, validating the histological classification of stages based on gonadal morphology. The expression patterns for many of these steroidogenic and sex determination and differentiation genes was strongly conserved between spotties and other species. Interestingly, expression of the masculinising gene amh (anti-Müllerian hormone) was upregulated at the onset of sex change, suggesting a potential early regulatory role of amh as a proximate trigger of sex change. Some major male-pathway (e.g. sox9a, znrf3) and female-pathway (e.g. cyp19a1a, rspo1) genes and also some epigenetic-related genes (e.g. kdm6bb) exhibited unexpected expression patterns based on mammalian and reptilian models. This suggests that the genetic cascade orchestrating sex change in spotty is regulated by a genetic network that is broadly conserved among protogynous sex-changing fish, albeit with some variations in the roles of specific genes. It also seems likely that epigenetic factors play an important role in the regulation of sex change. The role of cortisol as a potential trigger of sex change was tested through in vivo administration of cortisol (5000 µg) to female spotties for 71 days. Although this treatment did not induce sex change, upregulation of amh expression was observed, implying a physiological effect on its expression by cortisol, but this was obviously not strong enough to set off the full genetic cascade triggering sex change. Several technical issues, such as the relative rapid release of cortisol from the implants used (8 – 10 days) or the seasonality of this study potentially impacted the likelihood of sex change due to cortisol treatment. For the first time in a sequentially hermaphroditic fish, the patterns of expression for key steroidogenic and epigenetic regulatory genes were also evaluated in the head kidney, the organ functionally analogous to the adrenal gland in mammals and the site of most stress hormone production. Spotties implanted with cortisol, showed upregulation of hsd11b2 (11β-hydroxysteroid dehydrogenase type 2) expression confirming the physiological effect of cortisol previously observed. However, significant sex-biased expression of major sex-pathway genes was generally not observed in the head kidney of spotties socially induced to change sex. Finally, an in vitro culture system for fish ovaries was successfully developed, enabling research of the effect of E2, 11KT and cortisol in sex change to be investigated in spotties and potentially other sex changing fish. Oocyte degeneration associated with tissue culture was observed across treatments, but doses of E2 higher than 10 ng/mL were found to minimise this effect. Neither 11KT nor cortisol was observed to induce sex change of spotty ovaries. This outcome was hypothesised to be affected by the duration of the culture (i.e. 21 days), which was substantially shorter than the time needed for sex change to be completed in vivo (i.e. 70 days). Nonetheless, the valuable organ culture system developed here creates new opportunities to couple in vitro manipulative studies with promising techniques such as single-cell sequencing. Successful induction of sex change in captive spotties, the ability to precisely stage transitioning gonads, the development of a gene panel that accurately captures the ovary-to-testis genetic transition, and the establishment of an ovarian culture system reinforce the utility of spotty as an exceptional model for the study of fish sequential hermaphroditism. These experiments and resources set the baseline for further exploration of the role of stress during sex change at the level of both the gonad and the head kidney in this and other systems

Neuroendocrinology of Life History and Stress in Anemonefishes

A complete understanding of how the nervous and endocrine systems interplay within an organism is pivotal for the full characterization of the regulation of its physiological processes, as well as the interaction with its surroundings. These topics are now of major interest in the context of the existing threats linked with global change. In this chapter, we review the current knowledge regarding how anemonefishes respond and acclimate to environmental cues at the morphological, physiological, and behavioural levels. We focus on two natural life history events, metamorphosis and protandrous (male-to-female) sex change, both reflecting the extraordinary phenotypic plasticity of Amphiprioninae, and the potential ways in which the stress axis could mediate these transformations. Additionally, we highlight existing research on the consequences of ocean warming and acidification on these valuable teleost fishes. Finally, we synthesize the scarce data available to date in anemonefishes, an emerging biological model, together with knowledge acquired in this field using canonical fish species (e.g., zebrafish, medaka) or species of economic importance (e.g., salmonids, sea bass)

Subsurface chemical nanoidentification by nano-FTIR spectroscopy

Nano-FTIR spectroscopy allows chemical characterization of composite surfaces, but its capability in subsurface analysis is not much explored. The authors show that spectra from thin surface layers differ from those of subsurface layers of the same organic material, and establish a method for distinguishing them in experiments