Early life stage mechanisms of an active fish species to cope with ocean warming and hypoxia as interacting stressors.

Ocean's characteristics are rapidly changing, modifying environmental suitability for early life stages of fish. We assessed whether the chronic effects of warming (24 °C) and hypoxia (<2-2.5 mg L-1) will be amplified by the combination of these stressors on mortality, growth, behaviour, metabolism and oxidative stress of early stages of the white seabream Diplodus sargus. Combined warming and hypoxia synergistically increased larval mortality by >51%. Warming induced faster growth in length and slower gains in weight when compared to other treatments. Boldness and exploration were not directly affected, but swimming activity increased under all test treatments. Under the combination of warming and hypoxia, routine metabolic rate (RMR) significantly decreases when compared to other treatments and shows a negative thermal dependence. Superoxide dismutase and catalase activities increased under warming and were maintained similar to control levels under hypoxia or under combined stressors. Under hypoxia, the enzymatic activities were not enough to prevent oxidative damages as lipid peroxidation and DNA damage increased above control levels. Hypoxia reduced electron transport system activity (cellular respiration) and isocitrate dehydrogenase activity (aerobic metabolism) below control levels. However, lactate dehydrogenase activity (anaerobic metabolism) did not differ among treatments. A Redundancy Analysis showed that ∼99% of the variability in mortality, growth, behaviour and RMR among treatments can be explained by molecular responses. Mortality and growth are highly influenced by oxidative stress and energy metabolism, exhibiting a positive relationship with reactive oxygen species and a negative relationship with aerobic metabolism, regardless of treatment. Under hypoxic condition, RMR, boldness and swimming activity have a positive relationship with anaerobic metabolism regardless of temperature. Thus, seabreams may use anaerobic reliance to counterbalance the effects of the stressors on RMR, activity and growth. The outcomes suggests that early life stages of white seabream overcame the single and combined effects of hypoxia and warming.

Mating sounds in the two-spotted goby, Pomatoschistus flavescens: Effects of water temperature on acoustic featuresa).

Acoustic signals in teleost fishes play a fundamental role in reproduction. As fish are ectothermic animals, temperature has the potential to change their signal production and detection, with further implications for mating interactions. In this study, we describe the mating sounds made by the two-spotted goby, Pomatoschistus flavescens, for the first time and further investigate the effect of temperature on the acoustic features. Courtship sounds of 15 two-spotted goby males were recorded at three different temperatures: 16 °C, 19 °C, and 21 °C. As seen for other marine gobies, two-spotted goby produced two courtship sounds: drums and thumps. Drums showed similar acoustic features to other Pomatoschistus species already studied. Calling rates for both kinds of sound were not affected by the increases in temperature. However, pulse rate increased from 16 °C to 19 °C and stabilised between 19 °C and 21 °C, suggesting that two-spotted gobies reached their physiological limits at 19 °C. Spectral features were also affected by temperature, presenting higher values at 19 °C. Whether or not the observed changes in acoustic features with temperature lead to changes in mating remains to be addressed. Studies like the present one are fundamental to better comprehend how reproduction will be affected by global warming in soniferous fishes.

Effects of temperature on acoustic and visual courtship and reproductive success in the two-spotted goby Pomatoschistus flavescens.

Fish are ectothermic and small changes in water temperature could greatly affect reproduction. The two-spotted goby is a small semi-pelagic species that uses visual and acoustic displays to mate. Here, we studied the effect of temperature (16 and 20 °C) on acoustic and visual courtship and associated reproductive success in 39 males. Temperature influenced male visual courtship performed outside the nest, but it did not influence calling rate and the number of laid eggs. Interestingly, the number of sounds (drums) was the sole predictor of spawning success. These findings suggest that exposure to different temperatures within the species' natural range affect courtship behaviour but not its reproductive success. We propose that finding the link between acoustic behaviour and reproduction in fishes offers the opportunity to monitor fish sounds both in the lab and in nature to learn how they respond to environmental changes and human impacts, namely global warming.

Impact of anthropogenic noise on the survival and development of meagre (Argyrosomus regius) early life stages.

The growth of human populations has been driving an unprecedent and widespread increase in marine traffic, posing a real threat to marine biodiversity. Even though we are now aware of the negative effects of shipping noise exposure on fish, information about the impact on their early life stages continues to lack. Meagre (Argyrosomus regius) is a vocal fish that uses estuaries with high levels of anthropogenic noise pollution as both breeding areas and nurseries. Here, the effects of boat noise exposure on the development and survival of meagre larvae were studied. Embryos and larvae were exposed to either noise (boat noise playback) or control treatments (coils producing a similar electric field to the speakers) and hatching rate, survival rate, morphometric traits and stress-related biomarkers, at hatching and at 2 days-post-hatching (dph) were analyzed. Results showed no conclusive effects of the impact of boat noise playback, even though there was an increased lipid droplet consumption and a decrease in body depth at 2dph larvae under this stressor. The assessment of oxidative stress and energy metabolism-related biomarkers at hatching showed a marginal decrease in superoxide dismutase (SOD) activity and no changes in DNA damage or electron transport system activity (ETS), although it cannot be disregarded that those effects could only be visible at later stages of larval development. Whether these morphological and developmental results have implications in later stages remains to be investigated. Further studies with longer exposure and wild meagre could help deepen this knowledge and provide a better understanding of how anthropogenic noise can impact meagre early stages.

Hsp65-producing Lactococcus lactis inhibits experimental autoimmune encephalomyelitis by preventing cell migration into spinal cord.

Multiple sclerosis is an autoimmune disease that affects the central nervous system. Because of its complexity and the difficulty to treat, searching for immunoregulatory responses that reduce the clinical signs of disease by non-aggressive mechanisms and without adverse effects is a scientific challenge. Herein we propose a protocol of oral tolerance induction that prevented and controlled MOG-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. The genetically modified strain HSP65-producing Lactococcus lactis was orally administered for 5 consecutive days either before or during disease development in mice. Both protocols of feeding HSP65 resulted in significant reduction in the clinical score of EAE. Frequencies of LAP+CD4+Foxp3- regulatory T cells were higher in spleens and inguinal lymph nodes of fed mice. In addition, intravital microscopy showed that adherence of leukocytes to venules in the spinal cord was reduced in orally treated mice. Oral treatment with HSP65-producing L.lactis prevented leukocytes to leave the secondary lymphoid organs, therefore they could not reach the central nervous system. Despite the inhibition of pathological immune response that drive EAE development, activated T cells were at normal frequencies suggesting that oral tolerance did not induce general immunosuppression, but it led to specific control of pathogenic T cells. Our results indicate a novel therapeutic strategy to prevent and control autoimmune diseases such as multiple sclerosis.

GATA4 as a maestro of gut regionalization.

How gut regionalization impacts microbiota and immunity is unclear. In this issue of Immunity, Earley et al. show that jejumal GATA4 expression controls bacteria colonization through retinol metabolism and IgA production. This metabolic-immune axis limits intestinal Th17 responses and immunopathology.

The role of IgA in gastrointestinal helminthiasis: A systematic review.

Immunoglobulin-A (IgA) is an important mediator of immunity and has been associated with protection against several pathogens, although its role in gastrointestinal infections remains unclear. Then, the aim of this systematic review was to synthesize qualitative evidence in respect of IgA as mediator of protective immunity against gastrointestinal helminths. Following recommended guidelines, we searched for articles published between January 1990 and October 2019 that evaluated IgA levels and their association with gastrointestinal helminth infections. Twenty-five articles were included after screening 1,546 titles and abstracts, as well as reading in full 52 selected articles. Consistent associations between higher IgA levels and lower parasitological parameters were only found in mice, rats, and sheep. However, the role of IgA in other host species remains uncertain, making it difficult to create a consensus. Therefore, it is too soon to claim that IgA is an effective protective factor against gastrointestinal helminths, and further studies are still needed.

Effects of exposure to elevated temperature and different food levels on the escape response and metabolism of early life stages of white seabream, Diplodus sargus.

Recent literature suggests that anthropogenic stressors can disrupt ecologically relevant behaviours in fish, such as the ability to escape from predators. Disruption of these behaviours at critical life history transitions, such as the transition from the pelagic environment to the juvenile/adult habitat, may have even greater repercussions. The literature suggests that an increase in temperature can affect fish escape response, as well as metabolism; however, few studies have focused on the acute sensitivity responses and the potential for acclimation through developmental plasticity. Here, we aimed at evaluating the acute and long-term effects of exposure to warming conditions on the escape response and routine metabolic rate (RMR) of early life stages of the white seabream, Diplodus sargus. Additionally, as food availability may modulate the response to warming, we further tested the effects of long-term exposure to high temperature and food shortage, as individual and interacting drivers, on escape response and RMR. Temperature treatments were adjusted to ambient temperature (19°C) and a high temperature (22°C). Feeding treatments were established as high ration and low ration (50% of high ration). Escape response and RMR were measured after the high temperature was reached (acute exposure) and after 4 weeks (prolonged exposure). Acute warming had a significant effect on escape response and generated an upward trend in RMR. In the long term, however, there seems to be an acclimation of the escape response and RMR. Food shortage, interacting with high temperature, led to an increase in latency response and a significant reduction in RMR. The current study provides relevant experimental data on fishes' behavioural and physiological responses to the combined effects of multiple stressors. This knowledge can be incorporated in recruitment models, thereby contributing to fine-tuning of models required for fisheries management and species conservation.

Oral tolerance as antigen-specific immunotherapy.

Oral tolerance is a physiological phenomenon described more than a century ago as a suppressive immune response to antigens that gain access to the body by the oral route. It is a robust and long-lasting event with local and systemic effects in which the generation of mucosally induced regulatory T cells (iTreg) plays an essential role. The idea of using oral tolerance to inhibit autoimmune and allergic diseases by oral administration of target antigens was an important development that was successfully tested in 1980s. Since then, several studies have shown that feeding specific antigens can be used to prevent and control chronic inflammatory diseases in both animal models and clinically. Therefore, oral tolerance can be classified as an antigen-specific form of oral immunotherapy (OIT). In the light of novel findings on mechanisms, sites of induction and factors affecting oral tolerance, this review will focus on specific characteristics of oral tolerance induction and how they impact in its therapeutic application.

Elevated temperature, but not decreased pH, impairs reproduction in a temperate fish.

Fish reproductive success is linked to the ability of couples to mate and produce clutches that successfully hatch. Environmental stressors like high temperature and low pH can jeopardize this energetically costly process. In this study, exposure to high temperature and low pH was tested on a marine temperate species, Gobiusculus flavescens, to evaluate effects on reproductive performance. Breeding pairs were assigned to different temperatures (+ 0 °C, + 3 °C relative to in situ temperature) and pH levels (8.0, 7.6), in a cross-factorial design for a 3-month period. Reproduction activity, success, and paternal investment were measured throughout the exposure period. Results show reproduction is impaired by elevated temperature, while low pH had little impact. Breeding pairs under high temperature had 3% to 10% hatching success, up to 30% less eggs and eggs up to 20% smaller. Although paternal investment was not affected by tested parameters, males of breeding pairs exposed to elevated temperature had smaller gonadosomatic indexes, which might indicate a lack of investment in the reproductive process. Overall, results show that elevated temperature, expected more frequently in the near future, as a consequence of global warming, may impair key processes like reproduction in temperate fish, with potential consequences for fitness and population replenishment.

Compartmentalized gut lymph node drainage dictates adaptive immune responses.

The intestinal immune system has the challenging task of tolerating foreign nutrients and the commensal microbiome, while excluding or eliminating ingested pathogens. Failure of this balance leads to conditions such as inflammatory bowel diseases, food allergies and invasive gastrointestinal infections1. Multiple immune mechanisms are therefore in place to maintain tissue integrity, including balanced generation of effector T (TH) cells and FOXP3+ regulatory T (pTreg) cells, which mediate resistance to pathogens and regulate excessive immune activation, respectively1-4. The gut-draining lymph nodes (gLNs) are key sites for orchestrating adaptive immunity to luminal perturbations5-7. However, it is unclear how they simultaneously support tolerogenic and inflammatory reactions. Here we show that gLNs are immunologically specific to the functional gut segment that they drain. Stromal and dendritic cell gene signatures and polarization of T cells against the same luminal antigen differ between gLNs, with the proximal small intestine-draining gLNs preferentially giving rise to tolerogenic responses and the distal gLNs to pro-inflammatory T cell responses. This segregation permitted the targeting of distal gLNs for vaccination and the maintenance of duodenal pTreg cell induction during colonic infection. Conversely, the compartmentalized dichotomy was perturbed by surgical removal of select distal gLNs and duodenal infection, with effects on both lymphoid organ and tissue immune responses. Our findings reveal that the conflict between tolerogenic and inflammatory intestinal responses is in part resolved by discrete gLN drainage, and encourage antigen targeting to specific gut segments for therapeutic immune modulation.

Whey Protein Isolate-Supplemented Beverage, Fermented by Lactobacillus casei BL23 and Propionibacterium freudenreichii 138, in the Prevention of Mucositis in Mice.

Mucositis is a clinically important gastrointestinal inflammatory infirmity, generated by antineoplastic drugs cytotoxic effects. The inflammatory process caused by this disease frequently leads to derangements in the alimentary tract and great malaise for the patient. Novel strategies are necessary for its prevention or treatment, as currently available treatments of mucositis have several limitations in relieving its symptoms. In this context, several research groups have investigated the use of probiotic bacteria, and in particular dairy bacterial strains. Compelling evidences reveal that milk fermented by certain probiotic bacteria has the capacity to ameliorate intestinal inflammatory disorders. In addition, innovative probiotic delivery strategies, based on probiotics incorporation into protective matrices, such as whey proteins, were able to increase the therapeutic effect of probiotic strains by providing extra protection for bacteria against environmental stresses. Therefore, in this study, we evaluated the role of the whey protein isolate (WPI), when added to skim milk fermented by Lactobacillus casei BL23 (L. casei BL23) or by Propionibacterium freudenreichii CIRM-BIA138 (P. freudenreichii 138), as a protective matrix against in vitro stress challenges. In addition, we investigated the therapeutic effect of these fermented beverages in a murine model of mucositis induced by 5-Fluorouracil (5-FU). Our results demonstrated that milk supplementation with 30% (w/v) of WPI increases the survival rate of both strains when challenged with acid, bile salts, high temperature and cold storage stresses, compared to fermented skim milk without the addition of WPI. Moreover, treatment with the probiotic beverages prevented weight loss and intestinal damages in mice receiving 5-FU. We conclude that the presence of WPI maximizes the anti-inflammatory effects of L. casei BL23, but not for P. freudenreichii 138, suggesting that whey protein enhancement of probiotic activity might be strain-dependent.

Sand smelt ability to cope and recover from ocean's elevated CO2 levels.

Considered a major environmental concern, ocean acidification has induced a recent research boost into effects on marine biodiversity and possible ecological, physiological, and behavioural impacts. Although the majority of literature indicate negative effects of future acidification scenarios, most studies are conducted for just a few days or weeks, which may be insufficient to detect the capacity of an organism to adjust to environmental changes through phenotypic plasticity. Here, the effects and the capacity of sand smelt larvae Atherina presbyter to cope and recover (through a treatment combination strategy) from short (15 days) and long-term exposure (45 days) to increasing pCO2 levels (control: ~515 µatm, pH = 8.07; medium: ~940 µatm, pH = 7.84; high: ~1500 µatm, pH = 7.66) were measured, addressing larval development traits, behavioural lateralization, and biochemical biomarkers related with oxidative stress and damage, and energy metabolism and reserves. Although behavioural lateralization was not affected by high pCO2 exposure, morphometric changes, energetic costs, and oxidative stress damage were impacted differently through different exposures periods. Generally, short-time exposures led to different responses to either medium or high pCO2 levels (e.g. development, cellular metabolism, or damage), while on the long-term the response patterns tend to become similar between them, with both acidification scenarios inducing DNA damage and tending to lower growth rates. Additionally, when organisms were transferred to lower acidified condition, they were not able to recover from the mentioned DNA damage impacts. Overall, results suggest that exposure to future ocean acidification scenarios can induce sublethal effects on early life-stages of fish, but effects are dependent on duration of exposure, and are likely not reversible. Furthermore, to improve our understanding on species sensitivity and adaptation strategies, results reinforce the need to use multiple biological endpoints when assessing the effects of ocean acidification on marine organisms.

Trichoderma asperelloides Spores Downregulate dectin1/2 and TLR2 Receptors of Mice Macrophages and Decrease Candida parapsilosis Phagocytosis Independent of the M1/M2 Polarization.

The intensive use of pesticides to control pests in agriculture has promoted several issues relating to environment. As chemical pesticides remain controversial, biocontrol agents originating from fungi could be an alternative. Among them, we highlight biocontrol agents derived from the fungi genus Trichoderma, which have been documented in limiting the growth of other phytopathogenic fungus in the roots and leaves of several plant species. An important member of this genus is Trichoderma asperelloides, whose biocontrol agents have been used to promote plant growth while also treating soil diseases caused by microorganisms in both greenhouses and outdoor crops. To evaluate the safety of fungal biological agents for human health, tests to detect potentially adverse effects, such as allergenicity, toxicity, infectivity and pathogenicity, are crucial. In addition, identifying possible immunomodulating properties of fungal biocontrol agents merits further investigation. Thus, the aim of this study was to evaluate the effects of T. asperelloides spores in the internalization of Candida parapsilosis yeast by mice phagocytes, in order to elucidate the cellular and molecular mechanism of this interaction, as a model to understand possible in vivo effects of this fungus. For this, mice were exposed to a fungal spore suspension through-intraperitoneal injection, euthanized and cells from the peripheral blood and peritoneal cavity were collected for functional, quantitative and phenotypic analysis, throughout analysis of membrane receptors gene expression, phagocytosis ability and cells immunophenotyping M1 (CCR7 and CD86) and M2 (CCR2 and CD206). Our analyses showed that phagocytes exposed to fungal spores had reduced phagocytic capacity, as well as a decrease in the quantity of neutrophils and monocytes in the peripheral blood and peritoneal cavity. Moreover, macrophages exposed to T. asperelloides spores did not display the phenotypic profile M1/M2, and had reduced expression of pattern recognition receptors, such as TLR2, dectin-1 and dectin-2, all involved in the first line of defense against clinically important yeasts. Our data could infer that T. asperelloides spores may confer susceptibility to infection by C. parapsilosis.

Tissue adaptation: Implications for gut immunity and tolerance.

Tissue adaptation is an intrinsic component of immune cell development, influencing both resistance to pathogens and tolerance. Chronically stimulated surfaces of the body, in particular the gut mucosa, are the major sites where immune cells traffic and reside. Their adaptation to these environments requires constant discrimination between natural stimulation coming from harmless microbiota and food, and pathogens that need to be cleared. This review will focus on the adaptation of lymphocytes to the gut mucosa, a highly specialized environment that can help us understand the plasticity of leukocytes arriving at various tissue sites and how tissue-related factors operate to shape immune cell fate and function.

Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis.

BACKGROUND: Mucositis is one of the most relevant gastrointestinal inflammatory conditions in humans, generated by the use of chemotherapy drugs, such as 5-fluoracil (5-FU). 5-FU-induced mucositis affects 80% of patients undergoing oncological treatment causing mucosal gut dysfunctions and great discomfort. As current therapy drugs presents limitations in alleviating mucositis symptoms, alternative strategies are being pursued. Recent studies have shown that the antimicrobial pancreatitis-associated protein (PAP) has a protective role in intestinal inflammatory processes. Indeed, it was demonstrated that a recombinant strain of Lactococcus lactis expressing human PAP (LL-PAP) could prevent and improve murine DNBS-induced colitis, an inflammatory bowel disease (IBD) that causes severe inflammation of the colon. Hence, in this study we sought to evaluate the protective effects of LL-PAP on 5-FU-induced experimental mucositis in BALB/c mice as a novel approach to treat the disease. RESULTS: Our results show that non-recombinant L. lactis NZ9000 have antagonistic activity, in vitro, against the enteroinvasive gastrointestinal pathogen L. monocytogenes and confirmed PAP inhibitory effect against Opportunistic E. faecalis. Moreover, L. lactis was able to prevent histological damage, reduce neutrophil and eosinophil infiltration and secretory Immunoglobulin-A in mice injected with 5-FU. Recombinant lactococci carrying antimicrobial PAP did not improve those markers of inflammation, although its expression was associated with villous architecture preservation and increased secretory granules density inside Paneth cells in response to 5-FU inflammation. CONCLUSIONS: We have demonstrated for the first time that L. lactis NZ9000 by itself, is able to prevent 5-FU-induced intestinal inflammation in BALB/c mice. Moreover, PAP delivered by recombinant L. lactis strain showed additional protective effects in mice epithelium, revealing to be a promising strategy to treat intestinal mucositis.

Painted Goby Larvae under High-CO2 Fail to Recognize Reef Sounds.

Atmospheric CO2 levels have been increasing at an unprecedented rate due to anthropogenic activity. Consequently, ocean pCO2 is increasing and pH decreasing, affecting marine life, including fish. For many coastal marine fishes, selection of the adult habitat occurs at the end of the pelagic larval phase. Fish larvae use a range of sensory cues, including sound, for locating settlement habitat. This study tested the effect of elevated CO2 on the ability of settlement-stage temperate fish to use auditory cues from adult coastal reef habitats. Wild late larval stages of painted goby (Pomatoschistus pictus) were exposed to control pCO2 (532 µatm, pH 8.06) and high pCO2 (1503 µatm, pH 7.66) conditions, likely to occur in nearshore regions subjected to upwelling events by the end of the century, and tested in an auditory choice chamber for their preference or avoidance to nighttime reef recordings. Fish reared in control pCO2 conditions discriminated reef soundscapes and were attracted by reef recordings. This behaviour changed in fish reared in the high CO2 conditions, with settlement-stage larvae strongly avoiding reef recordings. This study provides evidence that ocean acidification might affect the auditory responses of larval stages of temperate reef fish species, with potentially significant impacts on their survival.

Benthic food webs support the production of sympatric flatfish larvae in estuarine nursery habitat.

Identifying nursery habitats is of paramount importance to define proper management and conservation strategies for flatfish species. Flatfish nursery studies usually report upon habitat occupation, but few attempted to quantify the importance of those habitats to larvae development. The reliance of two sympatric flatfish species larvae, the European flounder Platichthys flesus and the common sole Solea solea, on the estuarine food web (benthic vs. pelagic) was determined through carbon and nitrogen stable isotope analysis. The organic matter sources supporting the production of P. flesus and S. solea larvae biomass originates chiefly in the benthic food web. However, these species have significantly different δ13C and δ15N values which suggests that they prey on organisms that use a different mixture of sources or assimilate different components from similar OM pools (or both).

Effects of ocean acidification on the swimming ability, development and biochemical responses of sand smelt larvae.

Ocean acidification, recognized as a major threat to marine ecosystems, has developed into one of the fastest growing fields of research in marine sciences. Several studies on fish larval stages point to abnormal behaviours, malformations and increased mortality rates as a result of exposure to increased levels of CO2. However, other studies fail to recognize any consequence, suggesting species-specific sensitivity to increased levels of CO2, highlighting the need of further research. In this study we investigated the effects of exposure to elevated pCO2 on behaviour, development, oxidative stress and energy metabolism of sand smelt larvae, Atherina presbyter. Larvae were caught at Arrábida Marine Park (Portugal) and exposed to different pCO2 levels (control: ~600µatm, pH=8.03; medium: ~1000µatm, pH=7.85; high: ~1800µatm, pH=7.64) up to 15days, after which critical swimming speed (Ucrit), morphometric traits and biochemical biomarkers were determined. Measured biomarkers were related with: 1) oxidative stress - superoxide dismutase and catalase enzyme activities, levels of lipid peroxidation and DNA damage, and levels of superoxide anion production; 2) energy metabolism - total carbohydrate levels, electron transport system activity, lactate dehydrogenase and isocitrate dehydrogenase enzyme activities. Swimming speed was not affected by treatment, but exposure to increasing levels of pCO2 leads to higher energetic costs and morphometric changes, with larger larvae in high pCO2 treatment and smaller larvae in medium pCO2 treatment. The efficient antioxidant response capacity and increase in energetic metabolism only registered at the medium pCO2 treatment may indicate that at higher pCO2 levels the capacity of larvae to restore their internal balance can be impaired. Our findings illustrate the need of using multiple approaches to explore the consequences of future pCO2 levels on organisms.