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  • Modulation of intestinal growth and differentiation by photoperiod and dietary treatment during smoltification in Atlantic salmon (Salmo salar, L.)
    Publication . Fernandes Duarte, Vilma Vanessa; Gaetano, Pasqualina; Striberny, Anja; Hazlerigg, David; Jørgensen, Even H.; Fuentes, Juan; Campinho, Marco António
    Atlantic salmon undergo smoltification, a process that prepares the fish to enter and thrive in seawater (SW). Several physiological changes occur during smolting, especially in osmoregulatory tissues, the gill, the kidney, and the intestine. Here we characterized the effects on intestinal morphogenesis of two different, commonly used smoltification regimes during the end of the freshwater phase, photoperiod and/or the addition of salt and amino acid supplements in the diet. We focused on intestinal morphological differentiation, i.e., external perimeter, absorptive perimeter, tissue thickness, and villi density. In addition, we quantified cell proliferation (PCNA positive) and Na+, K+-ATPase (NKA) and Na+, K+,2Cl- (NKCCs) co-transporters expression and enterocyte apicobasal distribution by immunohistochemistry. These analyses show that the anterior and posterior intestines have different developmental dynamics during smoltification. In both intestinal regions, photoperiod and dietary treatment increased the absorptive perimeter. In addition, diet and photoperiod treatments differentially stimulated NKA protein expression in the anterior intestine. NKCC apical-basolateral expression in the enterocytes increased after SW entry in the anterior and posterior intestines. In conclusion, our results show that, as smoltification progresses, the anterior intestine responds more readily to experimental conditions than the posterior intestine. In our study, photoperiod and dietary treatment seem to enhance the development of the capacity to tolerate SW.
  • Macro and microscopic maturation stage key of green crab (Carcinus maenas, Linnaeus 1758): reproductive cycle and differences among estuarine systems
    Publication . Ovelheiro, Andreia; N. Monteiro, J.; Gonçalves, Patrícia; Campinho, Marco António; Maia, Francisco; Teodosio, Maria; Leitão, Francisco
    Gonadal histology is a helpful tool to validate species' macroscopic maturity stages in fisheries biology. Regardless of the many studies on Carcinus maenas, there are still few concerning gonads tissue histology and description of maturity stages. In Portugal, information regarding this species' biology to help support the regulation of crab fisheries, including the minimum landing size, has not yet been defined. In this work, the macro and microscopic characteristics of the reproductive cycle of C. maenas, oogenesis, and spermatogenesis development stages along the Portuguese coast are described, and a new macroscopic scale based on the histological analyses is suggested. During 2019 and 2020, adult C. maenas (both males and females) were collected from the Ria de Aveiro estuary, Rio Sado estuary, and Ria Formosa lagoon, respectively, North, Center, and South Portugal. No-significant differences (P > 0.05) were observed in carapace width and individual weight between all systems. Significant differences (P < 0.05) were observed in gonad weight, gonadosomatic index, and Fulton's condition index between the Rio Sado estuary and Ria Formosa lagoon. Furthermore, significant differences (P < 0.05) in the oocyte diameter between the northern and southernmost locations suggest a geographic variation related to the local environmental conditions of each system. Principal component analysis (PCA) revealed maturity stage and carapace width an association, and a similar morphometry between Ria de Aveiro and Ria Formosa. Temperature was correlated to both maturity stage and carapace width in Ria de Aveiro and Ria Formosa. From the histological analysis and based on microscopic criteria, it was observed that females previously classified within the late macroscopic development stage 2 should be considered mature, so a classification change in the current ovary's developmental stage is proposed. These findings can allow fisheries researchers to reclassify the estimations of maturity ogives and help support the regulation of this species' fishery. The proposed macroscopic scale was validated by histological analyses and can be used elsewhere.
  • Ioxynil and diethylstilbestrol increase the risks of cardiovascular and thyroid dysfunction in zebrafish
    Publication . Li, Yi-Feng; Rodrigues, Joana; Campinho, Marco António
    Endocrine disruption results from exposure to chemicals that alter the function of the endocrine system in animals. Chronic 60 days of exposure to a low dose (0.1 mu M) of ioxynil (IOX) or diethylstilbestrol (DES) via food was used to determine the effects of these chemicals on the physiology of the heart and thyroid follicles in juvenile zebrafish. Immunofluorescence analysis and subsequent 3D morphometric analysis of the zebrafish heart revealed that chronic exposure to IOX induced ventricle deformation and significant volume increase (p < 0.001). DES exposure caused a change in ventricle morphology, but volume was unaffected. Alongside, it was found that DES exposure upregulated endothelial related genes (angptl1b, mhc1lia, mybpc2a, ptgir, notch1b and vwf) involved in vascular homeostasis. Both IOX and DES exposure caused a change in thyroid follicle morphology. Notably, in IOX exposed juveniles, thyroid fol-licle hypertrophy was observed; and in DES-exposed fish, an enlarged thyroid field was present. In summary, chronic exposure of juvenile zebrafish to IOX and DES affected the heart and the thyroid. Given that both chemicals are able to change the morphology of the thyroid it indicates that they behave as endocrine disruptive chemicals (EDCs). Heart function dynamically changes thyroid morphology, and function and hence it is likely that the observed cardiac effects of IOX and DES are the source of altered thyroid status in these fish.
  • In a zebrafish biomedical model of human Allan-Herndon-Dudley syndrome impaired MTH signaling leads to decreased neural cell diversity
    Publication . Silva, Nadia; Campinho, Marco António
    Maternally derived thyroid hormone (T3) is a fundamental factor for vertebrate neurodevelopment. In humans, mutations on the thyroid hormones (TH) exclusive transporter monocarboxylic acid transporter 8 (MCT8) lead to the Allan-Herndon-Dudley syndrome (AHDS). Patients with AHDS present severe underdevelopment of the central nervous system, with profound cognitive and locomotor consequences. Functional impairment of zebrafish T3 exclusive membrane transporter Mct8 phenocopies many symptoms observed in patients with AHDS, thus providing an outstanding animal model to study this human condition. In addition, it was previously shown in the zebrafish mct8 KD model that maternal T3 (MTH) acts as an integrator of different key developmental pathways during zebrafish development. MethodsUsing a zebrafish Mct8 knockdown model, with consequent inhibition of maternal thyroid hormones (MTH) uptake to the target cells, we analyzed genes modulated by MTH by qPCR in a temporal series from the start of segmentation through hatching. Survival (TUNEL) and proliferation (PH3) of neural progenitor cells (dla, her2) were determined, and the cellular distribution of neural MTH-target genes in the spinal cord during development was characterized. In addition, in-vivo live imaging was performed to access NOTCH overexpression action on cell division in this AHDS model. We determined the developmental time window when MTH is required for appropriate CNS development in the zebrafish; MTH is not involved in neuroectoderm specification but is fundamental in the early stages of neurogenesis by promoting the maintenance of specific neural progenitor populations. MTH signaling is required for developing different neural cell types and maintaining spinal cord cytoarchitecture, and modulation of NOTCH signaling in a non-autonomous cell manner is involved in this process. DiscussionThe findings show that MTH allows the enrichment of neural progenitor pools, regulating the cell diversity output observed by the end of embryogenesis and that Mct8 impairment restricts CNS development. This work contributes to the understanding of the cellular mechanisms underlying human AHDS.
  • Editorial: the thyroid and Covid-19, volume II
    Publication . Jiménez, Javier; Campinho, Marco António; Nogueira, Celia Regina; Sgarbi, Jose
    Coronaviruses are enveloped RNA viruses of wide distribution in humans associated with mild respiratory disease. By contrast, severe acute respiratory syndrome coronavirus (SARS-CoV) is one of those coronaviruses that can cause fatal illness. In late December 2019, an outburst of pneumonia of unknown cause in Wuhan, China, was identified as the early stage of the coronavirus disease (COVID-19) pandemic outbreak, and the SARS-CoV-2 was found responsible (1). Two main proteins expressed by SARS-CoV-2 are essential for the manifestations of COVID-19. The first is the transmembrane protease serine 2 (TMPRSS2), which acts on the transcription and replication of the virus. The second is the Spike protein found on the surface of viral particles, which binds to angiotensin-converting enzyme 2 (ACE2) in tissue cells and is a determinant for transmitting infection. Therefore, SARS-CoV-2 infection depends on two steps: ACE2 receptor recognition via Spike protein and cell membrane fusion via transmembrane protease (2). ACE2 is expressed in different tissues, and the thyroid is no exception (3). It has been shown that the thyroid gland has high expression levels of ACE2, which may explain the direct effects on the thyroid parenchyma, making it more susceptible to viral attack (3). SARS-CoV-2 infection can lead to thyroid diseases by severely destroying parafollicular and follicular epithelial cells, leading to follicle rupture. As a result, SARS-CoV-2 virus infections are associated with inflammatory thyroid diseases such as subacute thyroiditis, Graves’ disease, thyrotoxicosis, Hashimoto’s thyroiditis, and euthyroid patient syndrome (4). As regards thyroid cancer, the COVID-19 pandemic has also affected its traditional management, and the consequences of this strategic change are largely unknown. Furthermore, it is intriguing that if the virus attacks the thyroid gland, it can also modulate thyroid cancer behavior (5).
  • Photoperiod and dietary treatment in freshwater modulate the short-term intestinal response to seawater in atlantic salmon (salmo salar)
    Publication . Gaetano, Pasqualina; Fernandes Duarte, Vilma Vanessa; Striberny, Anja; Hazlerigg, David; Jørgensen, Even H.; Campinho, Marco António; Fuentes, Juan
    Stimulation and timing of smoltification are essential for successful Atlantic salmon (Salmo salar) aquaculture. This study investigated intestinal responses during dietary and photoperiod manipulation in freshwater (FW) and after a subsequent seven days residence in seawater (SW). "Small" and "large" Atlantic salmon parr (-40 g and -130 g respectively) were treated in FW for 12 weeks and thereafter transferred to SW for seven days. During the FW phase, fish underwent two different light conditions, 24 L:0D - 24 L ("LL-LL" groups) and 7 L:17D - 24 L ("SP-LL" groups) or fed with either regular feed ("LL-LL C" and "SP-LL C" groups) or feed enriched with a salt mix plus free tryptophan ("LL-LL + diet" and "SP-LL + diet" groups). We analyzed Na+/K+-ATPase (NKA) activity, tissue bioelectrical properties in Ussing chambers, and intestinal fluid composition. The NKA activity showed minor variations in relation to fish size, treatments, or intestinal region (anterior or posterior). Photoperiod modulated epithelial bioelectrical properties (Isc and Rt) of the anterior and posterior intestine, particularly transepithelial resistance (Rt). Pharmacological experiments, targeting apical Na+/K+/2Cl- (NKCC2) and Na+/ Cl (NCC) co-transporters revealed intestinal region-and water salinity-dependent effects. In addition, stimu-lation of the intracellular cAMP with forskolin and IBMX showed intestinal region-, water salinity, and treatment-dependence responses with clear functional specialization of the anterior and posterior intestine. The intestinal fluid composition reflected the ability to process ingested SW and showed little variation in large fish. In sum-mary, our data suggest a better pre-adaptation of the intestine during light-stimulated smoltification (SP-LL groups), and the combination of light and diet might give, in an industrial aquaculture setting, an advantage to smaller, but not larger smolts. Intestinal fluid composition in small fish can be used as an index of intestinal function and may act as a long-term performance proxy in SW Atlantic salmon.
  • More than one way to smoltify a salmon? Effects of dietary and light treatment on smolt development and seawater growth performance in Atlantic salmon
    Publication . Striberny, Anja; Lauritzen, Daniel E.; Fuentes, Juan; Campinho, Marco António; Gaetano, Pasqualina; Duarte, Vilma; Hazlerigg, David G.; Jorgensen, Even H.
    Post-smolt mortality and stunted growth is a problem in Norwegian salmon farming that can be partly traced back to sub-optimal smolt quality in connection with ongoing changes in smolt production practices. In the present study, we compared smolt development and post-smolt performance in seawater (SW) of Atlantic salmon subjected to 1) traditional light treatment (short photoperiod (SP) long photoperiod (LL)), 2) a dietary treatment alone (LL-LL + diet), 3) a combination of light and diet (SP-LL + diet) treatment and 4) no stimulation (LL-LL control) during the freshwater (FW) phase. The transition diet consisted of feed supplemented with a salt mixture and the amino acid tryptophan. Effects of light and dietary treatment were tested on two different size classes (40 and 130 g) of parr. Response patterns to treatments were independent of initial size and findings discussed below apply to both. Fish in the continuous light groups (LL-LL and LL-LL + diet) had the highest mass gain during the FW phase while light treated fish had a reduction in condition factor indicative of a true smolting. Hypoosmoregulatory ability in May was increased by diet, but not by light. However, a strong and treatment independent decrease in gill NKA alpha 1a gene expression indicated a complete shift from a FWto a SW type gill Na+, K+-ATPase. Moreover, plasma Cl- concentration and osmolality after 7 days in SW in May were well within the range expected for fully SW adapted fish in all treatment groups. Despite good hypoosmoregulatory abilities in all fish, there were strong treatment dependent effects on their growth during the two month SW residence. SW growth was negatively correlated with growth and change in K during the last part of the FW phase. A markedly higher specific growth rate and appetite in the SP-LL group than in the LL-LL group was likely a result of the light stimulated endocrine changes that occurred during smolting. Dietary treatment seemed to compensate for the lack of light treatment since feed intake and growth in LL-LL + diet fish was comparable to that in the SP-LL treated smolts. Feed intake and growth in the SP-LL + diet groups was highest, giving support for a notion that light and diet act through separate and additive mechanisms.
  • Editorial: New insights in thyroid and Covid-19
    Publication . Sgarbi, Jose Augusto; Nogueira, Celia Regina; Brenta, Gabriela; Campinho, Marco António
    The Coronaviruses Disease 2019 (Covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is perhaps the most dramatic threat to human health since the Spanish flu in 1918. Almost 700 million cases and more than 6 million deaths have been reported worldwide by November 20, 2022 (1). The lung is the main affected organ, and the most critical clinical presentation has been characterized by interstitial pneumonia, acute respiratory distress syndrome, multiple organ failure, and death (2). Multiple endocrine organs, such as the pituitary, pancreas, adrenal, gonads, and thyroid gland, have also been affected (3). Detrimental effects on thyroid function have been reported in patients with and without pre-existing thyroid disease. Nonthyroidal illness syndrome (NTIS), subacute thyroiditis (SAT), Hashimoto’s thyroiditis, and Graves’ disease have been the most frequent thyroid dysfunctions associated with Covid-19 (4). Two major pathophysiological models have been implicated, a direct effect by virus attack causing follicular cells damage and an indirect effect caused by an immune-inflammatory abnormal response to the virus (5). Most recently, thyroid autoimmune diseases have also been reported following Covid-19 vaccination (6).
  • Editorial: the role of thyroid hormones in vertebrate development, volume II
    Publication . Campinho, Marco António; Sachs, Laurent M.
    The thyroid hormone is an important signaling molecule system involved in vertebrate development, acting at the embryonic and post-embryonic levels (1). Although this has long been established, a detailed understanding of the thyroid hormone’s developmental action still needs to be fully understood. The present Frontiers in Endocrinology Research Topic aims to close this knowledge gap by bringing together a collection of papers addressing what is known and the challenges ahead on the role of thyroid hormones in vertebrate development, from teleosts to humans. The published manuscripts highlight the pivotal action of thyroid hormones in vertebrate neurodevelopment (Lee at al.; Ng et al.; O’Shaughnessy et al.; Richard et al.; Silva and Campinho; Valcárcel-Hernández et al.), post natal development in amphibians (8–10) and how thyroid disorders can affect human development (Ru et al.; SeyedAlinaghi et al.).