Macropodus opercularis) has been a favored subject of behavioral research during the last decades of the 20th century. Lately, however, with a massively expanding genetic toolkit and a well annotated, fully sequenced genome, zebrafish (Danio rerio) became a central model of recent behavioral research. But, as the zebrafish behavioral repertoire is less complex than that of the paradise fish, the focus on zebrafish is a compromise. With the advent of novel methodologies, we think it is time to bring back paradise fish and develop it into a modern model of behavioral and evolutionary developmental biology (evo-devo) studies. The first step is to define the housing and husbandry conditions that can make a paradise fish a relevant and trustworthy model. Here, we define the relevant welfare parameters for keeping a healthy population of paradise fish and provide a detailed description of our recent experience in raising and successfully breeding this species under laboratory conditions.Paradise fish (Danio rerio) has become the dominant fish model species in biological and biomedical research. Multiple lines of evidence, however, suggest that the reliance on only a handful of genetic model organisms is problematic, as their unique evolutionary histories makes them less than ideal to study biological questions unrelated to their historically contingent adaptations. Therefore, a need has emerged to develop novel model species, better suited for studying particular problems. The paradise fish (Macropodus opercularis) has a much more complex behavioral repertoire than zebrafish and has been a favored model animal in ethological research during the last decades of the previous century. We believe that with currently available, easily adaptable genetic toolkits, this species could be easily developed into a popular model of behavioral genetics. Despite its earlier popularity, however, the description of a detailed housing and husbandry protocol for this species is still missing from scientific literature. We present here a detailed description of how to raise and breed paradise fish successfully under laboratory conditions, and also discuss some of the challenges we faced while creating a stable breeding population for this species in our facility.Thanks to its small size, external fertilization and fecundity, over the past four decades, zebrafish ( Macropodus opercularis  is a small sized (8–11 cm long), obligatory air-breathing, tropical freshwater fish species. It is native to Southeast Asia [Paradise fish, ast Asia ,3,4,5, wast Asia . FloatinThe species belongs to the group of anabantoids , most of which are freshwater fish native in Africa and Asia ,8,9. The2. Many anabantoid species are therefore obligatory air breathers and must surface to breath air to survive [The anabantoids are also known colloquially as “labyrinth fish” because they evolved a specialized organ that helps them to use the surface air for gas-exchange ,7,8,9. T survive . Air-breathing also influences a wide range of behaviors in the labyrinth fish, including territorial display, social communication, courtship, breeding and parental care ,8. TheseFoam nest-builders can build their nests either on the surface of the water or under water (depends on the species) . SurfaceDanio rerio) and medaka (Oryzias latipes)), replaced paradise fish in the realm of behavioral studies as well. This unique set of behaviors make the labyrinth fish ideally suited for a range of studies. Indeed, not so long ago, the paradise fish was a popular model for ethologists, and was studied for different aspects of its behavioral repertoire ,24,25,26M. opercularis was first introduced to Europe in 1869, where it became popular first in France, but soon it was available also in the UK and USA for hobby aquarists [quarists . Interesquarists ,3,4,5. Besides being “good practice” that helps experiments, optimal husbandry should also take into account that new research guidelines aim to cause minimal discomfort to the studied animals ,28. The M. opercularis into a modern model of behavioral studies, our primary goal is to establish the best husbandry and housing techniques for the species under scientific laboratory conditions. We present here a short assessment of our results and also discuss the challenges we encountered during this process, with the aim to present recommendations on how to start housing of this species in scientific laboratories. As we are interested to develop Paradise fish used in this study were maintained and bred in the animal facility of the Institute of Biology at ELTE Eötvös Loránd University. All experimental procedures were approved by the Hungarian National Food Chain Safety Office (Permit Number: PE/EA/406—7/2020). 4 (at 100 mg/L concentration for 2 min) followed by a NaClO (“bleach”) treatment (at 50 mg/L concentration for 5 min) and raised for 2–3 days in a 28.5 °C incubator. On the fourth day, the hatched and surviving larvae  were transferred to the fish facility.The fish were imported into the facility from a local breeder at embryonic developmental stages (less than ~10 h old). The imported embryos were disinfected with a wash of KMNOIn the facility, larvae were kept in 3 L plastic containers at the density of 20–40 individuals per container for the first month. The containers had no automated filtration, therefore water quality was maintained with daily cleaning and manual water changes. As environmental enrichment, we added a few biofilter pebbles into each container, which also helped maintaining water quality. Room temperature was kept between 24 and 28 °C, and photoperiod was set on a 13 h Light:11 h Dark cycle. Fish water was buffered to 350 µS conductivity and pH 7.5.Artemia salina nauplii larvae as live food. Larvae were fed thrice a day with a mixture of commercial tropical and zebrafish food , Sera Micron , SDS 100, SDS 200, SDS 300 ; Zebrafeed , TetraMin baby ). This diet has been supplemented with freshly hatched (24 h old) Approximately 40% of the larvae (70/180) survived to the age of 1.5–2 months, when they were moved into glass aquaria with bio-sponge filtration. Forty individuals were placed into a ~25 L aquarium (57 × 20 × 27 cm), whereas the remaining 30 individuals were placed to a ~20 L aquarium (50 × 20 × 25 cm). We continued to use biofilter pebbles as enrichment. When fish reached 3 months post-fertilization (mpf), we placed them into four larger glass aquariums (46 × 35 × 24 cm ~ 38.5 L) with approximately 31 L of actively used water volume in each. As only 65 individuals (derived from the imported embryos) survived to this age, the stocking density was about 15–17 individuals per aquarium. For additional environmental enrichment, we introduced small plastic floating plant leaves and green Styrofoam pieces A,B. Sexual dimorphism started to become apparent at ~5 mpf (adolescence) and was fully visible by 7–8 mpf C. At thi3, NO2− and NO3− levels were tested once a month by JBL test kits ). After 5 months, the feeding regime changed for twice daily feeding. To feed adult paradise fish, we used a mixture of commercial dry adult zebrafish and tropical fish food , supplemented with 48 h old, freshly hatched Artemia nauplii.Water parameters were kept as above and regularly tested , with a peak between May and July , with apFish breeding in our facility started when the animals were older than 6 months. Breeding occurred continuously from November to the end of summer. Breeding couples were rotated weekly and set up in 15 L aquariums (40 × 25 × 25 cm). To help the males with their nest-building activities, we provided in each breeding tank five small plastic floating plant leaves, five pieces of green Styrofoam (for bubble nest-building) and two pieces of plastic pipes . Fish were generally introduced to their breeding aquariums on Friday afternoons, and after an acclimatization period during the weekend, mating started at the beginning of the following week. Courtship and parental behaviors could be readily observed in these breeding pairs A.During the first year of housing, we encountered some diseased individuals in our breeding population and we also faced some difficulties with fish rearing. To improve housing conditions, we introduced a RO (re-osmosis water filter) unit  to our facility, and for our weekly water changes, we used water that was buffered to the desired parameters  from outer sump containers. To ensure the stability of pH in the holding tanks, we added sterilized coral crumbs and limestone pebbles into the bio-filter sponges and as substrate materials into the tanks.We also improved the environmental enrichment in the holding aquaria by introducing vertically floating green mosquito net pieces to imitate water plants  B. Coral After successfully rearing the first generation of paradise fish in our facility and stabilizing breeding, we also started testing different rearing conditions.In our first trials, we used either 10 L aquariums or 5 L plastic containers, both equipped with bio-sponge filter units. When using 10 L glass aquariums, we raised either 100 or 50 individuals in each of the 3 aquariums used in parallel. We performed this trial twice, using a total of 450 larvae. In case of the 5 L plastic containers, the trial was repeated 3 times, and for every trial, we placed 50–70 individuals in each of the 2 containers used in parallel. A total of 360 larvae have been tested under this condition.When we abandoned sponge filtration units and switched back to using standing water, we started using 3 L plastic containers. Using these conditions, different initial rearing densities have been tested in parallel holding designs. We compared the efficiencies of rearing 100 individuals per 3 L container , 70 larvae per 3 L container  and 50 individuals per 3 L container .Artemia nauplii as well, or they were given a mixture of nauplii and dry food immediately from 6 dpf.When testing for different diets, larvae were either fed exclusively with powdered dry food until the end of the third week, when we started to give them freshly hatched For all tested groups and conditions, individuals that reached the age of 12–13 wpf survived to adulthood.Observations of the group behaviors  were carried out in the holding aquariums before and after fish have been moved into and back from breeding tanks. Nest-building and courtship behavior could be observed in the breeding aquariums (see above). For a complete list of paradise fish behavioral elements see Diseased paradise fish were tested with both classical, bacteriological and pathological methods, and molecular (PCR) diagnostics. Bacteriology and pathological examinations were carried out by the Fish Pathology and Parasitology Team at the Institute for Veterinary Medical Research (IVMR) of the Centre for Agricultural Research (CAR). The autopsy included visual and microscopical observations of the fish after euthanasia. For bacteriological study, swab samples were collected from wounds and streaked on 10% sheep blood agar, Aeromonas agar, MacConkey agar and tryptic soy agar plates. In addition, direct isolation was performed from main inner organs  on the same media. The plates were incubated for 24–48 h at room temperature (average 20–22 °C). The colonies growing on agar plates were characterized by their appearances, visually and by light microscope, and identified taxonomically by molecular methods. In the preliminary examinations, the morphology and pigment production of the bacterial colonies were observed. In native smears, the shape and movement of each bacteria were checked by microscopy. Finally, selected unique bacterial colonies were subjected to universal 16S rDNA PCR and sequencing for taxonomical classification .Bacterial 16S rDNA was amplified from colonies by using the following primers: forward primer 5′-AGAGTTTGATCMTGGCTCAG-3′ and reverse primer 5′-GGTTACCTTGTTACGACTT-3′). The 25 µL PCR reactions were assembled using DreamTaq reagents , primers and template DNA. The PCR amplification program included 4 min of initial denaturation at 94 °C, then 35 cycles at 94 °C for 45 s, annealing at 55 °C for 60 s and at 72 °C for 45 s, and then 3 min of the final elongation at 72 °C, and was executed in a 2720 Thermal Cycler . The approximately 1400 bp large fragments were purified for direct sequencing using the Geneaid PCR purification Kit . Fragments were sequenced in both directions with the PCR primers using the BigDye Terminator Ready Reaction Mix v3.1. Cycle Sequencing Kit  according to the producer’s recommendations, and sequencing reactions were run on an ABI Prism 3100 Genetic Analyzer . Taxonomic identification homology searches were carried out in GenBank by using the Basic Local Alignment Search Tool (BLAST) algorithms on the National Center for Biotechnology Information (NCBI) server.Aeromonas hydrophila, Flavobacterium columnare, Mycobacterium spp., Pseudocapillaria tomentosa, Pseudoloma neurophilia and Pseudomonas aeruginosa. In the case of Mycobacterium spp. positivity, an additional mycobacteria-specific panel for the 6 most common aquarium species  was also used. The Circulum Kit sampling profile consists of high sensitivity, highly specificity and fast TaqMan qPCR and chemical assays, which are all validated by QM Diagnostics Company standards. In the sample preparation process, the company homogenizes the whole body of a fish in sterile phosphate-buffered saline buffer (PBS) and uses this sample for total nucleic acid purification. Swabs were vortexed in 1.5 mL tubes containing solution and then discarded with 175 μL of the sample supernatant being used for the DNA extraction. Sludge was diluted in sterile saline solution and the mixture was used for total nucleic acid purification.Molecular diagnostics was carried out with the Circulum Sampling Kit diagnostics package provided by QM Diagnostics . Following the recommendations of two earlier publications, describing health monitoring techniques in zebrafish facilities, we used the recommended environmental screening approaches for our sample collection ,37. SampFor certain body measurements or for some genetic/molecular experiments where tissue collection is needed (for example fin-clipping), the fish need to be anesthetized for shorter or longer periods of time. To perform these experiments, we adapted zebrafish fin-clipping protocols  for paraggplot2 package [Data visualization was performed in R  using th package . All figWe could not find any data in the literature about the survival rate of paradise fish embryos in their natural habitat, but with our initial conditions , approximately 35% (65/180) of the fertilized eggs reached adulthood.Further on, using the offspring of our founder population, we tested alternative conditions and observed that different stocking densities, feeding regimes and the type and size of the rearing tanks all affected survival rates . We starOnce we abandoned sponge filtration and started to rear paradise fish larvae at low larval stocking densities, in standing water bodies with smaller water volume and daily water changes, survival increased. Different rearing densities in 3 L plastic containers have been tested in parallel holding designs for 100, 70, 50 and 20–40 larvae per container. The results of all these trials are summarized in Artemia nauplii) from 6 dpf also helped us to increase the survival rate of larvae compared to batches where Artemia has just been added after 3 weeks post-fertilization (wpf) into the diet. After the first month, 48 h old Artemia nauplii could also be used for feeding. With these rearing conditions, we managed to raise 20–40% of fertilized embryos to adulthood  into the diet of larval fish increased the growth rate and survival rate of the fish ,44,45,46dulthood .Like other teleost, paradise fish undergo a period of metamorphosis while transitioning from larval to juvenile stages ,47. For After 6–7 wpf, juveniles were relocated into 10–15 L glass aquariums equipped with pumps and bio-sponge filters at the density of ~2 juveniles per 1 L of water. After an additional 6–8 weeks, young adults were relocated again (to aquariums with 31 L active capacity) to achieve lower stocking densities of ~1 fish per 2 L of water. Lower stocking densities were necessary for faster growth and lower aggression. Juvenile sibling fish were not aggressive with each other so they could be housed in larger groups. Once they reached sexual maturity and sexual dimorphism became apparent, however, fish displayed more aggressive behavior. We observed male–male, female–female and male–female fights, the number of which was considerably reduced when we decreased the stocking density. After 5–6 mpf, fish were kept in stable groups in their holding tanks, where the formation of hierarchies could be observed . Additional environmental enrichment elements and sheltering structures were introduced into the holding aquariums to help the fish to avoid aggressive individuals B. Using Our initial results suggested that water parameters and feeding regime optimized for zebrafish  were opt3, NO2− and NO3−.We also added frequently (monthly–bimonthly) nitrifying bacterial cultures to our tanks. These kept the biological filters stable in the aquariums, and we did not detect any harmful levels of NHAfter reaching adolescence (4–5 mpf), paradise fish started to develop their sexually dimorphic traits. Males elongated their dorsal, anal and caudal fins, while females developed rounder bellies. Coloration differences also became apparent between the sexes. Males displayed bright colors in a wide range of combinations (red or blue), depending on their behavior, while females usually lost some of their coloration to become pale-silvery. In parallel with the appearance of these sexually dimorphic traits A, fish aThese settled hierarchies, however, could be easily perturbed. Removing individual fish for short breeding sessions and later (after ~5 days) returning them to the holding tanks frequently resulted in the resumption of aggressive behavior and intense fights .Paradise fish are well-known for their ritualized display behaviors ,16,17,18We provide here a concise list and short description of the most common behaviors observed in our facility. Where possible, we try to break these behaviors down to their elements as previously described in the paradise fish ethogram. For a more complete list and more precise description of the behavioral elements, see It is a stereotypical, dominancy-related interaction which is highly costly for the individuals. Fish rotating their gill covers (opercula) generate hypoxic conditions in their body . In fullThese behaviors are common during courtship when they are usually performed simultaneously B. DuringOne fish bites another one. Biting is associated with highly aggressive situations .The dominant fish charges (CHA) and the fleeing individual escapes (ESC) with folded fins . Chasing can be short or prolonged, ending when the fleeing individual finds refuge, or the submissive display stops the aggression (CHA and or BIT) of the dominant fish.This posture is performed by females toward males or by submissive individuals toward dominant ones. The fins are folded, and the body may tilt vertically or horizontally. During mating, the lateral or vertical tilt usually changes into low-frequency tail beating vertical waggle (VEW) . As an aWhen threatening displays are not enough to establish a winner, physical fight ensues. During mouth-lock, two individuals bite and lock their mouths/jaws together, while waggling with their body . This caFor the majority of developmental experiments, it is important to collect the eggs right after fertilization. Due to the prolonged courtship, however, in paradise fish, it can be challenging to predict when this will happen. Furthermore, disturbing the fish or the nest too early can not only cause unnecessary stress to the animals, but it can also interfere with the breeding process. Therefore, we provide a concise description of the behavioral and physiological changes that can be observed during breeding and can used to foresee spawning events. During courtship, males perform a combination of the behavioral elements used for territorial defense and social display F–K. OperChanges in body color to a brighter complexion are usually the first sign that indicates the readiness of spawning in males. At the same time, males start to chase the females, occasionally performing opercular raise (OPE) or lateral spread display  in front of them. They also start to build nests by gulping bubbles of air into their mouth and eject them with a mucous coat under objects floating on the water surface (air-bubbling (A–B)) A. FemaleOnce the nest is ready, males will increase courtship towards receptive females. After performing lateral spread displays (LHH or LHT) B, they tDuring spawning, the male curves its body, to touch its tail, with the head at mid-section of the female while turning females upside-down in a close embrace—this posture is called anabantoid embrace (ANE) ,11 FiguJ. At thiFertilized and unfertilized eggs will float upwards into the bubble nest. Some eggs might miss the nest, but males will pick them up later by mouth (PIC) and relocate them into the bubble nest. Occasionally, females also try to help, but at this point, males can become aggressive and chase them away, until egg collection is finished. Individual spawning actions can be separated by several minutes. Males and females repeat these embracing circles in frequencies with sometimes not just spawning but pseudo-spawning actions as well.The whole mating process can last several hours (2–3 h on average) and by the end, the female releases approximately 400–600 eggs . These results are in line with the observations of Ward [Post-spawning males stay under the bubble nest and defend the territory around it vigorously. A set of nest-caring activities also ensues. Males continue to add new mucus-covered bubbles to the nest (A–B) and retrieve the embryos that escape from it (PIC). These activities continue until the hatched embryos are old enough to leave the bubble nest (~6 dpf).First signs of disease within our breeding colony appeared at 8 mpf (2 months after regular breeding has started). Affected individuals showed a variety of symptoms: scratches and open wounds on the body surface and inflammation around the mouth or cloacal area. Diseased individuals randomly appeared in different holding tanks, even after we applied strict quarantine for all the tanks, suggesting that we had to deal with an opportunistic pathogen and not an obligatory pathogenic microorganism.Interestingly, some symptoms were strictly sex-specific. For example, only males showed cloacal inflammation, initially the region around the anal papilla got red and sore, followed by a slow necrotization, resulting in the formation of a hole in the body wall A. After The other symptoms, red and inflamed mouth and inflamed scratches on the body surface, were typical for females B. OccasiAfter a period of investigation, we noted that only individuals used for breeding in the previous weeks tended to become sick. The incidence rate was higher among males than females. Our exploratory investigation also revealed that the pH of the holding tanks was fluctuating see . While fPseudomonas, Aerococcus and Comamonas, Aeromonas species were dominant.During the autopsy, no pathological changes or traces of internal or external parasite infection associated with the lesions were found. In the microbiological examination, a low number of slightly diverse bacteria could be isolated. Among them, next to Aeromonas hydrophila, Flavobacterium columnare, Mycobacterium spp., Pseudocapillaria tomentosa, Pseudoloma neurophilia and Pseudomonas aeruginosa. Using their PCR-based detection protocols, they could not detect any of the tested microorganisms in the mixed sludge sample from the holding tanks. The mixed sludge sample of the sick fish was positive for Aeromonas hydrophila and the mixed biofilm sample of the holding tanks for Mycobacterium spp. Moreover, the sick dead fish sample came back positive for Mycobacterium spp. and Aeromonas hydrophila as well.The Circulum sampling Kit service provided by QM Diagnostics company tests for the presence of the following agents: Mycobacterium spp.—positive samples were further tested by QM Diagnostics with their mycobacteria-specific PCR panel. This panel includes the six most common aquarium species . Although the biofilm sample got positive results with M. chelonae and M. fortuitum, in the specimen from sick dead fish, none of the 6 tested species could be detected, suggesting the presence of any additional mycobacterial species in the sick individuals and in the holding tanks.The Standard fish work required the adaption of anesthesia and fin-clipping protocols for paradise fish. In our trials, Tricaine at 120 mg/L concentration provided a safe and effective dose: all individuals became anesthetized within 3–5 min, depending on their body weight.We noticed that the pigmentation of both males and females became darker as they lost control during the anesthesia, suggesting an active, continuous control of melanophore morphology. Interestingly, this was different from the change in coloration under stress, when the fish tended to become pale. After the performing of desired procedures , the anesthetized individuals were immediately placed into recovery tanks to avoid desiccation. Fish recovered fully within 3–5 min and they swam to the surface to gasp air several times. They also toned down their bright body coloration and after 4–5 min they started to behave normally.All our fish  subjected to anesthesia survived without suffering any lasting harm.Over the past decades, the enhanced/intensive application in the research of zebrafish and to a lesser extent medaka as dominant genetic model organisms has established fish as useful tools to study the etiology of human diseases and to search for novel drug precursor molecules ,53,54,55Thanks to its complex behavioral repertoire, in the 1970s, paradise fish emerged as a leading model for behavioral research ,58,59,60Fish are poikilothermic aquatic animals who inhabit a complex three-dimensional environment which directly influences their welfare . Water c3) [In order to ensure optimal conditions, aquarium water should be continuously filtered or, if that is not an available option, should be changed on a regular basis . A filtr3) . 3—containing shells and corals, directly into the filter medium [Our initial routine included weekly changes of the water in the holding tanks from a buffered water supply . We soon realized, however, that after 3–4 days, a rapid acidification of the water occurred, and pH could drop as low as 5.8 by the end of the week. This occurred because biological filtration  combined with the accumulation of waste products excreted by the fish, which gradually induce the acidification of the water . This cor medium . As thesIn tropical regions, where paradise fish are endemic, temperature and the length of the days are the main reproductive stimuli for fish and the normal breeding season lasts from May to October . While oWe also made an effort to provide sufficient environmental enrichment in our holding tanks. Unfortunately, as research laboratory set-ups are generally designed to standardize conditions, they are too often stimulus-poor, barren tanks/aquaria ,66. SeveAs paradise fish get older and larger, lower stocking densities are required for ideal growth and reduced aggression. Thus, while for juveniles 30–40 fish per 20 L of water was a usable stocking density, for adults, we had to decrease stocking densities to approximately 10–12 fish per 30 L. With the addition of extra structural elements to the tank , the group quickly established a hierarchy and fish lived together without excessive stress. Typical nest-builder fish, such as the paradise fish, also need floating plant-like materials to create their nests ,8,9,14. Artemia nauplii larvae) is essential for the larvae, as in their absence they grow slower and die at higher rates. Other studies came to similar conclusions for other anabantoids [The rearing of anabantoid species has been shown to be a challenging task ,15, and bantoids ,29,45,46Betta splendens), for example, the inclusion of carotenoids in the diet improved both the body colors and the immune response as well [It is likely that including additional nutritional supplements, such as carotenoids, in their diet, could further improve the wellbeing of paradise fish stocks. Carotenoids for example can be important for pigment synthesis, but they also serve as a source of antioxidants and can modulate the immune response. Vertebrates are not able to synthetize carotenoids, so they have to uptake them through their food . In the  as well ,68. Once we established our housing and husbandry routines, we were also able to regularly observe some species-specific behavioral patterns. The development of the LO  not only helped anabantoids to adapt to extreme environmental conditions, but also resulted in the evolution of unique physiological and behavioral features . They al2 consumption, courtship and spawning action could be physiologically costly for paradise fish males .Body color could also provide information about the physiological condition. Paradise fish can dynamically change their body coloration and adapt it to their behavior. These changes are more profound in males, especially during courtship (using intense red coloration), which might also provide an explanation as to why males often get sick post-mating: if carotenoid reserves in the body are depleted during breeding, fish might be prone for diseases as a robust immune response might also necessitate carotenoids, just as in closely related bettas (trade-off between red coloration and immune response) ,68. We hAeromonas hydrophilia) in the wounds of ill fish. Finally, our observations also provided some insights into the emergence of disease in paradise fish. The random appearance of symptoms in unrelated individuals in different tanks suggested that an opportunistic pathogen/s could be behind the diseases. As immune suppression is known to lower the resistance to disease ,64,69, wParadise fish with their complex behavioral repertoire ,24,25,26Here, we presented a summary of our efforts that led to a stable breeding population of paradise fish in the animal facility of ELTE Eötvös Loránd University. Our results demonstrated that appropriately adjusted water pH, feeding regime and stocking densities during larval rearing and adult housing can significantly improve the conditions and survival rate of this species under laboratory conditions. We are confident, therefore, that the improved housing conditions and husbandry routines outlined above meet the general fish welfare guidelines  and will


Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants’ responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota.Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper ( Plants are metaorganisms that host a complex and dynamic consortium of bacteria, fungi, archaea and protists that communicate with plants by exchanging chemical signals throughout the rhizosphere and endosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning . TherefoArabidopsis, enhanced growth and photosynthesis promoted by air application of fungal VCs is associated with increases in levels of active forms of cytokinins (CKs), photosynthetic pigments and transitory starch in leaves, together with reductions in abscisic acid (ABA) contents, and changes in the transcriptome and proteome through mechanisms involving signaling of redox-activated photosynthesis and long-distance communication between roots and the aerial part of the plant  with molecular masses of less than 300 Da that act as efficient semiochemicals in interkingdom communication, participating in countless interactions among plants and microorganisms, and exerting a strong effect on microbial communities . VCs emihe plant .Trichoderma harzianum and two fungal phytopathogens  here we conducted studies to address the question of whether soil application of cell-free filtrates of beneficial and pathogenic fungal cultures can improve growth and yield of a plant species of agronomic interest  cultured under greenhouse and open field conditions, and whether this response is mediated by mechanisms comparable to those triggered by air application of fungal VCs. In addition, using amplicon sequencing targeting of the fungal ITS and bacterial 16S rRNA genes, we investigated whether filtrates of fungal cultures are capable of altering the indigenous soil microbial communities. Furthermore, using distillates (DEs) obtained from the fungal cultures, we evaluated the contribution made by fungal organic VCs (VOCs) in these responses. Our findings show that soil application of cell-free filtrates of beneficial and pathogenic fungi promotes root growth and enhances yield of pepper plants at least partially through mechanisms wherein activation of beneficial soil microbiota promoted by VOCs occurring in the fungal culture filtrates (CFs) could play important roles.To date, studies on the response of plants to soil application of cell-free filtrates from cultures of beneficial microorganisms have been centered on the effect of these compounds on plant growth and yield, but have not explored their modes/mechanisms of action in plants and/or their effects on resident soil microbial communities . Using tTrichoderma harzianum (CECT 2413), A. alternata (CECT 20912) and P. aurantiogriseum (CECT 20226) from glycerol stocks were inoculated into 500 mL Erlenmeyer flasks containing 200 mL liquid Murashige and Skoog (MS) medium supplemented with 90 mM sucrose. Cultures were incubated at 28°C with constant shaking at 180 rpm on a rotatory shaker for 7 days. Next, 100 mL of the cultures were poured into 2 L Erlenmeyer flasks containing 1 L MS-sucrose medium and incubated at 28°C with constant shaking at 180 rpm for 3 days, after which the fungal mycelium was removed using Whatman #1 filter paper. The resulting filtrate was sterilized using a 0.22 μm Millipore membrane filter and stored at 4°C whilst awaiting further use. DEs were obtained by distilling the CFs at 50°C using a R3000 (BUCHI) rotavapor following manufacturer’s instructions1.–1 m–2), 25°C /8 h dark, 15°C cycles. For greenhouse trials, plants were transplanted to 6 L pots filled with peat:sand:vermiculite mixture (1:1:1) and cultured for 7 weeks with natural light under 16 h 25°C /8 h 15°C conditions. Treatments with fungal CFs and DEs started when the plants developed their first two true leaves. Increasing volumes  of CFs and DEs were sequentially applied on soil once a week over 7 weeks. Each treatment was applied to twelve plants. Controls for DE-treated plants were plants irrigated with distilled water. Controls for CF-treated plants were plants irrigated with diluted (1:3) Murashige and Skoog (MS) medium supplemented with 1.5 mM each of glucose and fructose, which is the approximate concentration of both sugars in the three CFs. At the selected sampling times, leaves and commercial fruits were harvested and immediately ground to a fine powder in liquid nitrogen, then stored at −80°C prior to further biochemical characterization. “Commercial” Sweet Italian pepper fruits are defined as those having a minimum length of 14 cm.This work was carried out using Sweet Italian and Piquillo pepper plants cultured under greenhouse and field conditions, respectively. Seeds were sown and cultured in the dark at 24°C on filter paper moistened with distilled water. Ten days after sowing, the pepper seedlings were transplanted into small pots (250 mL) filled with peat:sand:vermiculite mixture (1:1:1) and cultured for 4 weeks in growth chambers providing 16 h light , plants were drip-irrigated with a 1:25 dilution of the original CF extract  twice: one month after transplanting and at flowering. Using the same method, control plants were irrigated with a 1:25 dilution of the MS medium supplemented with 1.5 mM each of glucose and fructose stock (10.4 L/plot). Commercial fruits were harvested on three occasions: first, third and fourth week of September. Those destined for compositional analyses were immediately ground to a fine powder in liquid nitrogen and stored at −80°C. “Commercial” Piquillo pepper fruits are defined as those having 9–10 cm length, 40–45 cm width, 40–45 g weight and 1.5–3 mm pericarp thickness.Field trials were conducted at the INTIA’s experimental station located near Sartaguda  between May and September of 2016 and 2018. Plants were transplanted from 250 mL pots (see above) to 22.40 mSoluble sugars in CFs and frozen powders of leaves and fruits were determined by HPLC with pulsed amperometric detection on a IC3000 Dionex system, as described in Volatile organic compounds in CFs and DEs were determined by gas chromatography-mass spectrometry (GC-MS) using the solid-phase microextraction (SPME) technique as described by Fruit hardness was determined using a penetrometer , by measuring the depth or rate of penetration of a rod driven into the fruit with a known force.–2 s–1, as described by 2 assimilation (An) and stomatal conductance (gs) were calculated as described by i) was calculated as the ratio of An to gs as described by Gas exchange rates were determined at 25°C with a photosynthetic photon flux density of 350 μmol m2 suggested that sequencing depth was sufficient to cover most of the detectable bacterial and fungal OTUs as the curves began to plateau or reached their asymptote for all sample types by treatment  using soil from the 250 mL pots with plants cultured for 4 weeks in growth chambers (see above). Three days after the third treatment with fungal CFs or DEs, three replicate samples each consisting of all the soil from three pots were collected, mixed, sieved to a < 2 mm particle size, lyophilized and stored at −80°C. Soil DNA was extracted from 10 grams of soil of each replicate using a DNeasy Mericon kit from QIAGEN. The quantity and purity of DNA were examined with a NanoDrop ND-1000 spectrophotometer . The bacterial 16S rRNA and fungal ITS libraries were constructed following the “16S metagenomics sequencing library preparation” protocol given for the Illumina MiSeq system, and using optimized primers targeting the hypervariable V3 and V4 regions of the 16S rRNA gene for the bacterial profile  and primreatment . OTUs wet-test using SPSS software. Differences were considered significant if P < 0.05. Both microbiome α- and β-diversity statistical analyses were performed using the R package “Phyloseq” v1.23.0  grown under greenhouse conditions to soil application of CFs from t growth , inducedt growth  and enhat growth . Furthert growth . Soil apt growth  or fruitt growth . Soil apn leaves . In addin leaves . Moreoven leaves . Consistreatment .We also characterized the responses of pepper plants (cv. Piquillo) grown in the field to soil application of the three fungal CFs. In two independent experiments, these treatments enhanced the number of commercial fruits per plant and yield  and alteP. aurantiogriseum CFs contained very much higher levels of CKs than those of A. alternata and T. harzianum  than those of A. alternata and T. harzianum . Moreover, ABA content in T. harzianum CF (20.1 ± 5.5 pmol/L) was higher than in A. alternata and P. aurantiogriseum CFs . Analyses of the organic volatilomes of the three CFs identified a total of 80 VOCs , Paraburkholderia (P. paradisi and P. silvatlantica), Burkholderia (B. arboris), Rhodoferax, Rhodanobacter (R. glycinis), and Massilia (M. varians), and reduced populations of Cellulosimicrobium (C. funkei)  , 4A. Soi funkei) . At spec indexes . At the CoA plot , indicatengisoli , 4A, 6, tiformis , 4A.Geotrichum (G. candidum), Candida (C. subhashii), Trichoderma (T. hamatum) and Myxocephala , and reduced those of Saccharomyces (S. cerevisiae)  . At specevisiae) . Howeverevisiae) . The CF  albida, , 8, and rmentans , 7.Here, we showed that soil application of CFs of beneficial and pathogenic fungi promotes root growth, accelerates fruit development, and increases the yield of commercial fruits of pepper plants cultured under greenhouse and field conditions. The differences observed between non-induced plants of different independent experiments could be ascribed to non-controlled environmental factors, such as differences in temperature and light intensity and quality. Therefore, CFs of both beneficial and phytopathogenic fungi can be used as biostimulants to improve crop yield. Because CFs alter fruit composition, they can potentially be used to improve fruit quality.A. alternata and P. aurantiogriseum CFs was surprising as it is well known that many fungal phytopathogens release secondary metabolites and proteins in their culture media that display phytotoxicity in tests on tissue culture germinating seeds, plantlets, calli and cellular suspensions . Therefore, the contrasting differences between our results and those of previous studies can be ascribed to the fact that, under the experimental conditions used in this study, A. alternata and P. aurantiogriseum are not active with respect to phytotoxin production.Root growth stimulation and enhanced fruit yield promoted by soil application of pensions . Phytotopensions . Here, wHere, we showed that the vast majority of VOCs released by fungi to aqueous culture media can be “rescued” by distillation, and analyzed the effect of soil application of these compounds in plants. This is, to our knowledge, the first study of the response of plants to soil application of VOCs distilled from microbial cultures. Under greenhouse conditions, the responses of pepper plants to soil application of the three DEs were comparable to those of CF-treated plants. Thus, in our experimental system, VOCs appear to be strong determinants of the plants’ responses to fungal CFs. Unlike air VC application, soil DE application did not promote shoot growth and photosynthesis, did not enhance WUEi and the contents of photosynthetic pigments, starch and soluble sugars in leaves, and did not reduce leaf ABA content, indicating that the mechanisms involved in the responses of plants to soil and air applications of volatiles are different.cis-thujopsene and α-longipinene, as they have been shown to promote shoot growth and root developmental changes when exogenously supplied to plants  have been shown to act as plant growth promoters that solubilize mineral phosphate, fix nitrogen, produce siderophores, synthesize hormones  and/or antagonize microbial phytopathogens -isomenthol; citronellol; α-bisabolol and β-selinene. It is evident that further efforts will be necessary to characterize the action potentials of these compounds on beneficial microbial populations.Ten compounds , acetic acid, 2-heptanone-4-methyl, 2-heptanone-4,6-dimethyl and 2-nonanone) are present in the three fungal DEs. It is likely that, individually or collectively, these compounds play important roles in the VOC-mediated activation of the beneficial microbiota in CF-treated soils. It is noteworthy that the response of the soil microbial community to Results presented in this work show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants’ responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota. Data provided here should motivate the applications of translational biology needed to reinforce agricultural practices with more natural, cheaper and sustainable approaches.The discovery that soil application of VOCs from both beneficial and pathogenic microorganisms can enhance root growth and yield and promote similar changes in the soil microbial communities extends knowledge of the diversity and complexity of the interactions involved in modulation of the physiology of the plant and its interaction with surrounding microbes, raising questions regarding the evolution of the processes, their ecological significance and potential applications. Because microbes respond to microbial VOCs , we propMicrobes and their volatiles contribute in the various aspects of biotic stress management. The release of volatiles by beneficial microbes directly protects plants by inhibiting growth of pathogenic organisms, and indirectly elicit plant defense response by enhancing immune response against pathogen attack . It has We show that soil application of cell-free filtrates of beneficial and pathogenic fungi promotes root growth and enhances yield of pepper plants through mechanisms wherein activation of beneficial soil microbiota by VOCs occurring in the fungal culture filtrates could play important roles.PRJNA748689.The original contributions presented in the study are publicly available. This data can be found here: National Center for Biotechnology Information (NCBI) BioProject database under accession number EB-F and JP-R designed the experiments, analyzed the data, and wrote the article with contributions from all authors. EB-F, GA, ÁS-L, AB, SG-A, ND, KD, and FJM performed most of the experiments. ECS performed the microbiome statistical analyses. JP-R supervised the experiments and conceived the project and research plans. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Psammomys obesus). Voluntary exercise strengthened general activity rhythms, improved memory and lowered anxiety- and depressive-like behaviors, enhanced oral glucose tolerance, and decreased plasma insulin levels and liver weight. Animals with access to a running wheel had larger heart weight and heart/body weight ratio, and thicker left ventricular wall. Our results demonstrate that exercising ameliorates pathological-like daily rhythms in activity and blood glucose levels, glucose tolerance and depressive- and anxiety-like behaviors in the sand rat model, supporting the important role of physical activity in modulating the “circadian syndrome” and circadian rhythm-related diseases. We suggest that the utilization of a diurnal rodent animal model may offer an effective way to further explore metabolic, cardiovascular, and affective-like behavioral changes related to chronodisruption and their underlying mechanisms.Emerging evidence suggests that disruption of circadian rhythmicity contributes to development of comorbid depression, cardiovascular diseases (CVD), and type 2 diabetes mellitus (T2DM). Physical exercise synchronizes the circadian system and has ameliorating effects on the depression- and anxiety-like phenotype induced by circadian disruption in mice and sand rats. We explored the beneficial effects of voluntary wheel running on daily rhythms, and the development of depression, T2DM, and CVD in a diurnal animal model, the fat sand rat ( Emerging evidence suggests that disruption of circadian rhythmicity may contribute to the development of these comorbidities, and we have proposed the term "circadian syndrome" for this combined pathological condition6.The comorbidity between depression, cardiovascular diseases (CVD), and type 2 diabetes mellitus (T2DM) has been repeatedly described in the literature”) that is re-set (entrained) through daily environmental timing cues, especially light but also feeding and physical activity, allowing living things to anticipate periodic daily events, to orchestrate internal temporal programs of behavioral and physiological functions, and to flexibly set the order and scheduling of such functions to optimize fitness in the real world. In mammals, a “central” or “master” clock in the hypothalamus of the brain (suprachiasmatic nucleus [SCN]) synchronizes “peripheral” clocks found in cells, tissues, and organs throughout the brain and body to regulate metabolism and temporal physiology8. This network of clocks is capable of adaptively re-aligning its oscillatory components under changing conditions, including to signals such as, e.g., temperature, blood glucose and oxygen levels, and glucocorticoids. Importantly, there is increasing recognition that pathological misalignment of network components—between the environment, behavior, SCN, and peripheral clocks  —has critical implications not only for an individual’s health and performance but also for the pathophysiology of aging and disease12. Such circadian disruption in animal models, as well as in humans, may lead to the “circadian syndrome,” with depressive-like behavior13, CVD9, and T2DM13.Circadian rhythms are the manifestation of an internal timekeeping system (the “circadian clockPsammomys obesus). Sand rats are diurnally active in nature14, but when kept indoors under standard laboratory conditions they demonstrate an unstable, nocturnal phase preference, with low amplitude, and in some cases, no rhythm at all16. Interestingly, similar findings have been reported for almost all diurnal species examined to date22; conversely, such circadian disruption under normal laboratory conditions has not been documented in nocturnal rodents22. The laboratory-associated disruption of circadian rhythmicity in diurnal rodents is even more pronounced when animals are kept under a short photoperiod regimen (5 h light:19 h dark)23. Within approximately 8 weeks of short photoperiod acclimation with standard rodent diet, sand rats develop the "circadian syndrome," manifesting as glucose intolerance, elevated plasma insulin levels, cataracts, CVD, and depressive- and anxiety-like behaviors6. Strong entrainment of the circadian system by keeping sand rats outdoors, where a wealth of biotic and abiotic variables cycle, or by bright light treatment, prevents the development of these disorders24.We have been investigating this syndrome in a unique animal model, the fat sand rat  = 4.91, p = 0.027]  were all arrhythmic  [12/12 arrhythmic], whereas sand rats kept with running heels were either diurnal , nocturnal , or arrhythmic (3/10) . Wheels sand rats demonstrated a more pronounced rhythm, with significant differences between all ZT points   Fig. .Figure 4The presence of a wheel in the cage had no significant effect on the development of cataracts . Nevertheless, 4/12 of the No wheels animals had mature cataracts versus none in the Wheels group (0/10).There was no effect of wheel presence in the cage on body weight at the end of the experiment (week 11) .The heart weight and heart/body weight ratio were larger in Wheels animals compared to No wheels animals   Fig. . No diffMeasuring anxiety-like behavior in the EPM, we found that animals maintained without running wheels spent less time in the open arms of the EPM than animals maintained with running wheels, as demonstrated by lower open/total time ratio measure  Fig. a. There The Wheels group showed a longer time to sink in the FST with near significant effect in the first sink  and a significant effect in the second sink  Fig. c.To examine the possibility that outcomes were influenced by the level of general activity rather than wheel effects on circadian rhythmicity, we computed the mean total activity per 24 h for animals maintained with wheels, and analyzed possible correlations between this measure and the behavioral and physiological measures described above. For the behavioral measures there were no significant correlations between activity and EPM open/closed ratio , FST sink 2 , or NORT discrimination ratio . For the physiological measures, there was only one significant correlation, between activity and glucose levels  but no significant correlations with insulin levels , heart/body weight ratio  or liver weight .Our results show that voluntary wheel running for 11 weeks inhibits the development of the “circadian syndrome” in fat sand rats housed indoors in standard laboratory conditions. Daily rhythms of general locomotor activity and blood glucose are strengthened, blood glucose and plasma insulin levels are lowered, oral glucose tolerance is enhanced, and tests of depressive- and anxiety-like behaviors and memory are improved.25 found that activity patterns of sand rats were more robust and less fragmented when they had free access to a running wheel, and the effect was more pronounced in animals acclimated to a short photoperiod. Of note, physical exercise elevates the arterial CO2 tension (PaCO2)33, and Adamovich et al. (2019)34 found that changes in carbon dioxide levels altered the expression of several core circadian clock genes and shifted circadian phase in cultured cells. We speculate that one mechanism by which voluntary exercise could influence the circadian system might be through the modulation of PaCO2.We found that sand rats kept without access to a running wheel were all behaviorally arrhythmic, whereas most sand rats kept with a running wheel showed a significant daily locomotor activity rhythm 7/10) and were either diurnal (5/10) or nocturnal (2/10), while only 3/10 were arrhythmic 24 found that sand rats subjected to morning bright light treatment, which enhances circadian rhythmicity24, showed robust daily rhythms in clock gene expression and behavior, and were normoglycemic, with higher glucose tolerance than the control group.Voluntary wheel running had a significant effect on baseline blood glucose levels and on glucose tolerance, with the No wheels group showing significantly higher blood glucose levels than the Wheels group, both at baseline and 120 min after oral glucose administration in the oral glucose tolerance test Fig. . Accordi35. At the beginning of the activity/feeding phase, when blood glucose is mainly of dietary origin35, glucose tolerance and blood glucose levels are at their highest36. During the resting/fasting period, glucose is recruited from endogenous gluconeogenesis in the liver, and glucose tolerance and blood glucose levels are lower36. Daily rhythmicity in blood glucose levels is regulated directly by the SCN and by peripheral clocks in the liver, pancreas, muscle, and white adipose tissue35. When the SCN is lesioned in rats, the daily fluctuation in glucose uptake and insulin sensitivity disappears36. Furthermore, mutations of clock genes cause disruption of glucose homeostasis, with severely disturbed daily blood glucose rhythms, hyperglycemia, and hypoinsulinemia38. However, rhythms in glucose tolerance and in blood glucose levels also emanate from behavioral rhythms in exercise and feeding40. Skeletal muscle is the major tissue responsible for insulin-mediated glucose utilization. The plasma membrane GLUT4 content is correlated with glucose transport activity in both animal models and human skeletal muscle41. Regular exercise results in elevated insulin- and contraction-stimulated glucose transport capacity by augmenting skeletal muscle GLUT4 levels42, and GLUT4 activity demonstrates circadian rhythmicity with higher expression levels and translocation during the activity phase than the rest phase43. In addition, feeding behavior modulates daily rhythms of blood glucose and glucagon concentrations40. The classic function of glucagon is to increase hepatic glucose output when glucose concentrations decline during fasting40. Ad libitum–fed rats show peaks in glucagon concentrations during the resting/fasting phase, shortly before the onset of the active/feeding phase40. In contrast, rats that start fasting at the onset of their activity phase have decreased plasma glucagon concentrations at the end of the resting period, instead of the expected increase40. Thus, further experiments will be required to determine the relative contributions of the SCN, peripheral clocks, and behavioral rhythms of exercise and feeding in accounting for the differences between the Wheels and the No wheels sand rats in glucose tolerance and blood glucose rhythmicity.Blood glucose homeostasis is manifested by daily rhythmicity in glucose tolerance and in blood glucose levels20 reporting the development of mature cataracts in animals kept under short photoperiod conditions, we did not find a significant effect of wheel running on the development of mature cataracts . This difference might be explained by the different length of the two experiments: Bilu et al. (2019)15 kept the sand rats under short photoperiod conditions for 20 weeks, whereas the current experiment lasted only 11 weeks, a time which might not be sufficient for the development of mature cataracts in these animals.In contrast to our previous study15, and that animals with diabetes had a larger heart weight than non-diabetic sand rats5. We ascribed these results to the pathological cardiovascular effects of hyperglycemia, such as elevated aortic stiffness and reduced myocardial metabolism, which alters diastolic function in patients with diabetes mellitus. Furthermore, higher fasting blood glucose levels and glycated hemoglobin are associated with abnormal left ventricular relaxation in people with diabetes44. However, in the current study, the heart weight and heart/body weight ratio were larger, and the left ventricular wall was thicker, in the Wheels group and not in the diabetic No wheels group  parietal thickness, without impairment of contractile strength or LV performance45. However, in order to differentiate between a purely physiological, adaptive process vs. a pathological hypertrophic cardiomyopathy, further experiments will be needed to examine aspects of the sand rat heart's anatomical and functional characteristics, such as LV cavity size, LV diastolic function, and the absence of T-wave inversion on electrocardiography.Another contrast with previous studies was the difference in heart weight, heart/body weight, and left ventricular wall thickness between the groups. In earlier studies we found that short photoperiod acclimated sand rats had a larger heart weight and heart/body weight ratio than neutral photoperiod acclimated sand rats46. During periods of hyperglycemia, glucose freely enters the hepatocytes, driving glycogen synthesis, and eventually causes the accumulation of excessive amounts of glycogen in the hepatocytes (glycogenosis), causing hepatomegaly and elevated aminotransferases. These abnormalities are readily reversible with sustained euglycemic control47. The other major cause of hepatomegaly in people with diabetes is steatosis. This is a function of the body habitus and state of insulin resistance, rather than glycemic control, and it may progress to fibrosis and cirrhosis47. In order to distinguish between these two causes of hepatomegaly, histological studies will be needed to assess for swollen hepatocytes containing excess glycogen in the cytoplasm, and often also in the nucleus. These are indicative of glycogenosis48. There was no significant difference in liver/body weight between the groups.The higher liver weight in the No wheels group compared to the Wheels group Fig.  is consi49. Cross-sectional studies associate high self-reported levels of regular exercise with improved mental health and low depression50. Moreover, self-reported high levels of habitual physical activity correlate with fewer symptoms of both anxiety and depression and better mental health51. The neurobiological effects of exercise, acting as a mood elevating agent, appear to influence several neural mechanisms related to depression and anxiety51. There is evidence that physical activity causes physiological changes in monoamine levels52, upregulates neurotrophic factors53, alters the levels of the stress hormone cortisol54, and leads to adaptations in limbic structures implicated in depressive and anxiety disorders.There has long been discussion regarding the ameliorating effects of exercise on cognitive functions and affect. However, exercise has only recently received the attention of the scientific community with the key interest in its effects on cognitive functions, affective disorders, spatial learning and memory, and as a non-drug method to maintain brain health55, including adrenaline and serotonin56. Antidepressants and mood stabilizers affect systems related to circadian rhythmicity57, and modifications of genes related to circadian rhythms influence affective-like behaviours. However, the mechanisms underlying the relationship between circadian rhythmicity and affective disorders are still not yet fully understood. In the present experiment, we found that animals maintained without running wheels showed higher anxiety- and depressive-like behaviors compared to animals maintained with running wheels  were used as subjects. Animals were individually housed in standard plastic cages (30 cm ′ 40 cm ′ 40 cm), in temperature-controlled rooms (25 °C)15. After 3 weeks of acclimation, the sand rats were divided into two groups, based on weight and blood glucose levels to avoid a baseline bias, and kept under short photoperiod : 12 sand rats were kept in cages with running wheels ("Wheels"), and 12 sand rats were kept in cages with no running wheels ("No wheels"). All animals were provided with ad-lib tap water and standard rodent food . Body weight was measured weekly during the experiment. In-cage general locomotor activity was monitored throughout the experiment, using IR motion detectors . Running activity in the wheels was recorded using inductive sensors . Data were collected at 6-min intervals using designated software 15. Wheel running was detected as activity in the IR motion detectors. All experimental procedures followed the NIH guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Tel Aviv University (permit number L15055). The study is reported in accordance with ARRIVE guidelines.24 male sand rats (68). On week 11 the sand rats were euthanized around ZT 7 (during the dark phase), and left ventricle wall thickness and heart and liver weights were measured.Before the onset of the experiments, all animals were maintained on a low-energy diet  and 12 h light:12 h dark cycle (lights on at 09:00 and off at 21:00), to prevent diabetes. All animals were weighed and tested for glucose tolerance before the start of the experiment. Animals were assigned to the experimental groups based on weight and blood glucose levels to avoid a baseline bias: 12 males in the Wheels group and 12 males in the No wheels group. Body weight was measured weekly during the experiment. On week 8 animals were evaluated in three standard behavioral tests of anxiety-like behavior , memory  and depression-like behavior . On week 9, around ZT 2, animals were weighed, blood was collected from the tail tips for glucose measurement , and oral glucose tolerance tests were performed. The plasma samples of 10 animals from each group were assayed for insulin. On week 10, all animals were tested for a 24-h blood glucose rhythm. Three days later, the animals’ eyes were examined for the presence of cataracts . Habituation took place by exposing the animal to the experimental apparatus one time for 5 min in the absence of objects, on the day before training. During the training phase sand rats were placed in the experimental apparatus in the presence of two identical objects and allowed to explore for 15 min. After a retention interval of 24 h, animals were placed again in the apparatus, where this time one of the objects was replaced by a novel one. Sand rats were allowed to explore for 15 min. Preference for the novel object was expressed as the percent time spent exploring the novel object relative to the total time spent exploring both objects. The objects were a glass bottle and a rectangular plastic box, both with approximately the same height. The identity of the objects, which one was novel or familiar, as well as the spatial location (whether the novel object was placed on the left or right side during the test session) of each object was balanced between groups. A preference for either object was not observed in this study. Each group's ability to recognize the novel object was determined by dividing the mean time of the animal exploring the novel object by the mean of the total time exploring the novel and familiar objects during the test session (Tnovel/[Tnovel + Tfamiliar]). In both tasks, objects were rinsed with ethanol between trials and before the first trial. All testing and training sessions were videotaped and analyzed by an experimenter blind to the treatment of the animals. It was considered exploration of the objects when animals were facing and sniffing the objects within very close proximity and/or touching15.Performed at ZT2, as described in Bilu et al. 201915.Performed in week 9, at ZT2, in animals fasted for 4 h, as described in Bilu et al. 2019Plasma insulin protein was assayed with an immunoassay ELISA kit  on blood collected in week 9, at ZT2.On week 10, all animals were tested for a 24-h blood glucose rhythm by collecting blood from the tail tip at ZT2, ZT8, ZT14 and ZT20 and measuring blood glucose levels using a glucometer .15.Performed on week 10, as described in Bilu et al. 201915. Subsequently, a section midway between the base and the apex, perpendicular to the longitudinal axis of the ventricle, was obtained to measure the average wall thickness of the free wall. Five equally spaced measurements of the left ventricular free wall were collected, and their values were averaged. Then, the liver was removed and rinsed in two washes of ice-cold saline. Major blood vessels and connective tissue were removed, the liver was blotted dry, and weighed.On week 11, the sand rats were euthanized, the heart was collected as described in Bilu et al. 201915.Statistical analysis was performed using Statistica 13.0 . Data were analyzed using students’ t-tests or analysis of variance (ANOVA) as appropriate, and statistical significance was accepted at p < 0.05. Correlations were evaluated using Pearson’s Correlations. The actograms and the significance of the daily rhythm in general locomotor activity was calculated by χ2 test using CTools 7.0 software by van der Veen on data collected on week 10. Activity pattern was defined as diurnal if more than 50% of activity occurred during the light phase, and nocturnal if more than 50% of activity occurred during the dark phase. To identify the existence of daily rhythms in blood glucose levels we used one-way ANOVAs with ZT as main factor within each group to analyze differences between levels at different ZT points. Significant ANOVA results were followed by LSD post-hoc test. A rhythm was considered significant when there was a significant difference between at least two ZT time points


What is the risk of recall and high-risk recall for devices undergoing US Food and Drug Administration (FDA) 510(k) clearance compared with premarket approval (PMA)?In this cohort study using the FDA’s 510(k) and PMA medical device database, 28 556 devices were reviewed. Although 97% of recalled devices had received 510(k) clearance, devices with PMA had 2.7 times the hazard of recall and 7.3 times the hazard of high-risk recall compared with devices with 510(k) clearance.This study suggests that, despite the requirement of clinical trials, high-risk devices approved via PMA were associated with greater safety concerns than previously reported; in addition, most recalls are for 510(k) devices, raising safety issues. The US Food and Drug Administration (FDA) uses 510(k) clearance and premarket approval (PMA) pathways to ensure device safety before marketing. Premarket approval evaluates high-risk medical devices and requires clinical trials, whereas 510(k) clearance evaluates moderate-risk devices and relies on benchtop  and descriptive data. Existing literature suggests that the clinical trials required by PMA are associated with reduced risk of recall compared with devices granted 510(k) clearance. Several investigators have found weaknesses in pivotal PMA trials, raising safety concerns. Furthermore, methodological factors may have led to a previous underestimation of recall risk for devices with PMA.To compare risk of recall and high-risk recall between devices that received 510(k) clearance and those that received PMA and to compare the risk of recall between devices for medical specialties.This cohort study compared devices with 510(k) clearance vs those with PMA that reached the market between January 1, 2008, and December 31, 2017. Two- to 12-year follow-up was obtained from the FDA’s 510(k) and PMA medical device database. Orthopedic surgery was chosen arbitrarily as the reference category for analysis between specialties because no baseline exists. Statistical analysis was performed from February 1 to November 1, 2020.The FDA issues recalls for safety concerns. These recalls are stratified into class I, II, and III, with class I representing high-risk issues for serious harm or death. The main outcome was the hazard ratio of any recall and class I recall between devices with PMA and those with 510(k) clearance. The secondary outcome was the recall hazard ratio between specialties with respect to the reference category. A single Cox proportional hazards regression model evaluating the association of medical specialty and FDA approval pathway with the risk of recall was performed.P < .001) and a hazard ratio for high-risk recall of 7.30 . Only radiologic devices were associated with an increased risk of recall , whereas 6 specialties were assocated with a decreased risk compared with the orthopedic reference category: general and plastic surgery, otolaryngology, obstetrics and gynecology, physical medicine, hematology, and general hospital.During the study period, 28 246 devices received 510(k) clearance and 310 devices (10.7%) received PMA; 3012 devices (10.7%) with 510(k) clearance and 84 devices (27.1%) with PMA were recalled. A total of 216 devices (0.8%) with 510(k) clearance and 16 devices (5.2%) with PMA had class I recalls. Devices with PMA compared with those with 510(k) clearance had a hazard ratio for recall of 2.74  clearance, also raising safety concerns. Strengthening postmarketing surveillance strategies and pivotal trials may improve device safety. This cohort study compares risk of recall and high-risk recall between medical devices that received US Food and Drug Administration 510(k) clearance and those that received premarket approval. Several highly publicized device failures have renewed calls for regulatory change.14 In brief, the FDA stratifies devices according to risk: class I is minimal , class II is moderate , and class III is high . The pathway that a novel device takes through the FDA to reach the market depends largely on this classification. Class III devices require a PMA, whereas class II devices typically undergo 510(k) clearance. Class I products are usually exempt from formal testing.15Medical device regulation has been described previously.18A fundamental difference between PMA and 510(k) clearance is in the burden of proof required. A successful PMA application must prove a reasonable assurance of safety and effectiveness, whereas the 510(k) application must show only substantial equivalence to another device already on the market. To achieve the former requires a clinical trial, whereas the latter frequently relies on benchtop  tests and descriptive analysis.19 found that only 27% of the studies used to support cardiovascular devices with PMA were randomized, and just 14% were blinded in any way. Barker et al20 reported similar concerns regarding study strength in pivotal trials for orthopedic devices. Despite these findings, most authors reporting on device safety have expressed greater concerns regarding 510(k) clearance because of the frequent lack of clinical evidence.23 In a study by Zuckerman et al,7 71% of high-risk recalls were for devices with 510(k) clearance. Day et al6 found that devices with 510(k) clearance were 11.5 times more likely to be recalled than those with PMA, whereas Somberg et al11 reported that devices with 510(k) clearance were twice as likely as those with PMA to have a high-risk recall. This trend has been demonstrated in the obstetric, otolaryngologic, and radiologic literature.12 In 2011, the Institute of Medicine completed an evaluation of the 510(k) clearance process over its 35 years and recommended it be phased out.22Investigators have found reason for concern in both approval pathways. Dhruva et al11 have attempted to account for some of these issues; however, methodological concerns still hamper the applicability of their results. Day et al6 looked only at recalls within the top 20 companies, which they divided into various medical specialties based on the supposed field the company most commonly serves. For instance, in their analysis, all Depuy and Medtronic devices were considered “orthopedics,” demonstrating the high level of subjectivity in this approach. Somberg et al11 considered only high-risk recalls and did not account for the time each device was on the market.To our knowledge, no peer-reviewed literature exists that accurately establishes the risk of recall for a device approved by 510(k) clearance or PMA. Nearly all of the existing literature retrospectively analyzes only the subset of recalled devices. This approach is problematic for multiple reasons: (1) the volume of devices with 510(k) clearance on the market is vastly greater than that of devices with PMA, so one would expect proportionately more recalls of devices with 510(k) clearance; (2) previous studies have not accounted for the duration of time the device is on the market and exposed to the risk of recall; and (3) devices can be recalled multiple times, so a single problematic device can disproportionately weigh on the results. Two authorsThe present study represents the largest to date, to our knowledge, that analyzes both all recalls and high-risk recalls. The goal is to establish an accurate assessment of the risk of recall and high-risk recall for devices with 510(k) clearance and those with PMA by performing a time-to-event analysis and to subsequently test the hypothesis that devices with 510(k) clearance pose a greater risk of recall than those with PMA. Secondary aims included comparing the risk of recall between medical specialties and evaluating the total number of recall events to address the impact of devices with multiple recalls.24 The filter option “Panel” was set to a specific FDA medical specialty  to classify each device into 1 of 19 specialties. This classification yielded 29 898 devices; however, a duplicate check in Excel found 1652 devices listed under 2 separate specialty panels. These devices were individually searched on the database using their FDA-given 510(k) number and then assigned to the specialty designated by the FDA as “510(k) Review Panel,” leaving a total of 28 246 unique devices with 510(k) clearance. This cohort study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline. The University of Missouri–Kansas City institutional review board deemed this study exempt from institutional review board approval because it does not involve human participants.From the FDA’s 510(k) database, all devices that received clearance between January 1, 2008, and December 31, 2017, were downloaded to a Microsoft Excel, version 16.0 (Microsoft Corp) spreadsheet.25 Manufacturers can submit PMA supplements for devices with existing PMA for several reasons, including labeling changes, device modifications, and expanding indications. Supplements do not exist for devices with 510(k) clearance, and significant changes require a new 510(k) application.26 Although valid criticisms have been raised about the safety of PMA supplements and how their accumulation over time can substantially alter the original device, the FDA does not treat supplements as unique devices, considering them only modifications to the original device.28 As such, in the database, a recall is listed only under the original device’s PMA number, even if the cause was from a change introduced by a supplement, as was the case with the Medtronic Sprint Fidelis Lead.30 To stay consistent with FDA methods, we included only the original PMA in the accounting of total devices with PMA.The PMA database was similarly queried, but with adding the filter “Supplement Type” set to “Originals Only.”31 Recalls can be issued for multiple parts of the same device; to prevent overcounting, recalls on the same date were counted as 1 recall.The end point of the study was December 31, 2019, providing 2 to 12 years of follow-up for the devices on the market. Owing to the size of the data set, data were abstracted repeatedly throughout January 2020 by clicking on the link for each device in the results from the searches described. Further relevant information for recalled devices, including recall dates and type, was then collected from the link for each individual device. The FDA codifies recalls according to the severity of potential harm, with class I being the highest, class II being moderate, and class III being the lowest.P values and 95% CIs for comparing each device specialty with orthopedics have been adjusted for multiple comparisons using the Bonferroni correction. All tests were 2-sided using an α of .05. A second Cox proportional hazards regression model was created to compare high-risk recalls (class I) between devices with PMA and devices with 510(k) clearance.Statistical analysis was performed from February 1 to November 1, 2020. Microsoft Excel, version 16.0 was used for descriptive analysis, and R, version 4.0.0  was used for statistical analysis. A single Cox proportional hazards regression model that evaluated the association of medical specialty and FDA approval pathway with the risk of recall was performed. Although selection of a reference category for the analysis between specialties is arbitrary because no true baseline exists, orthopedic surgery was chosen because the category contained the largest number of devices, and the overall percentage of recalled devices appeared similar to the group of all devices as a whole. The The Cox proportional hazards regression assumption was examined using Schoenfeld residuals and the complementary log-log plot. Several very minor departures from proportional hazards were seen, but these departures were limited to very early or very late time ranges.The FDA cleared a mean of 2825 devices  per year by 510(k) compared with a mean of 31 devices  approved per year under PMA. A total of 28 556 devices came to market during the 10-year study period; while the proportion of devices with PMA did increase from 0.7% to 1.5%, overall, only 310 devices (1.1%) were approved through the PMA process, and 28 246 devices received 510(k) clearance . There wThere were 5362 recall events among all devices included in the study. Of these, 97.3% (5218) were for devices with 510(k) approval, and 2.7% (144) were for PMA devices. The greater number of total recalls compared with devices recalled is secondary to the frequency of devices undergoing multiple recalls. Of the 84 devices with PMA that were recalled, 31.0% (26) had multiple recall events. Similarly, of the 3012 devices with 510(k) clearance that were recalled, 31.9% (960) experienced multiple recalls.P < .001). For only class I recalls, the hazard ratio for devices with PMA increased to 7.30  (Compared with devices with 510(k) clearance, the hazard ratio for recall of devices with PMA was 2.74  A and B. The specialty with the most devices was orthopedics (n = 5399), for which 24 devices were approved by PMA. Cardiovascular and radiology constituted the next largest specialty, with 3900 and 3577 devices, respectively. Cardiology was the specialty with the largest number of recalled devices with PMA (n = 115), with microbiology having the second largest number (n = 41).P < .001). Six specialties had significantly lower recall than the reference category: general and plastic surgery (8.4% [284 of 3368]), otolaryngology (5.1% [19 of 370]), obstetrics and gynecology (4.4% [31 of 708]), physical medicine (2.6% [16 of 607]), hematology (6.2% [33 of 529]), and general hospital (8.4% [224 of 2673])  demonstrated a significantly increased risk of recall relative to the reference category , with a hazard ratio of 1.57  .12 To our knowledge, this is the largest study to date to analyze both all recalls and high-risk recalls, with 28 556 devices analyzed over 10 years. It is also the first study, to our knowledge, to account for the duration a device is on the market using a time-to-event analysis. These factors likely contributed to rejecting the hypothesis that devices with 510(k) clearance pose a greater risk of recall than those with PMA. Instead, we found that devices with PMA have 2.7 times the hazard of any recall and 7.3 times the hazard of class I recall when compared with devices with 510(k) clearance. At 9 years after device approval, the risk of any recall for devices with PMA and 510(k) clearance is 32% vs 13% and decreases to about 6% vs 1%, respectively, when considering only class I recalls. However, devices with 510(k) clearance comprise 99% of the devices to reach the market and comprised approximately 97% of both the 3096 devices recalled and the 5362 total number of recalls identified in the study period, making them a significant source of safety concern despite the devices demonstrating a lower risk of recall.Ensuring the safety of medical devices is integral to public health. Multiple authors have attempted to assess medical device safety by investigating recalls.11 Similar findings are reported in the otolaryngology, ophthalmology, obstetrics and gynecology, and radiology literature.12In hindsight, this finding may appear intuitive, considering that devices with PMA are high-risk devices by definition and are more commonly subject to postapproval studies and surveillance. Overall, however, the existing literature supports the opposite conclusion, with estimates ranging from twice to nearly 12 times the risk of recall for devices with 510(k) clearance compared with those with PMA.20 Rather, the discrepancy between the present study and other studies may be attributable to 2 major differences in methods. First, most previous literature analyzed the total number of recalls in a given period, not accounting for vastly larger numbers of devices with 510(k) clearance, time on the market, or devices having multiple recalls. Second, several authors likely included PMA supplements in their analysis, although they did not report it in their methods.11 Supplements have been shown to often rely on no, or sometimes weak, clinical data for approval, and their accumulation over time, reported to range from a median of 6.5 to 50 supplements per device, may lead to essentially unique devices from the original.29 The FDA, however, considers supplements only as modifications, and including them in a recall analysis dilutes the risk of recall by overestimating the actual number of devices with PMA.29 This finding may explain why Somberg et al11 reported nearly 6000 devices with PMA in 9 years with a 0.45% probability of class I recall compared with the present study, which found 310 devices in 10 years with a 6% risk of class I recall. Supplements may increase the risk of device recall; however, that research is beyond the scope of this investigation.Most authors attribute their results to the clinical data required by the PMA; however, this runs counter to several studies showing significant shortcomings in the methods used for PMA pivotal trials.32 investigated recall between specialties over a 14-year period and found similar variations, albeit mainly between different fields. They also identified physical medicine and obstetrics and gynecology devices as having very low risk of recall, although their analysis included only high-risk recalls. Other investigators have attempted to compare recall between fields as well but with varied results.11 The disparate findings between studies are likely attributable to the methodological differences noted previously.With regard to secondary end points, we found significant variation in the risk of recall between specialties. Although only radiology demonstrated a significantly increased risk of recall, 6 specialties had a significantly lower risk of recall than the reference category (orthopedic surgery). It is possible that the different proportions of high-risk devices within each specialty may have contributed to these findings. For instance, both physical medicine and hematology had no devices with PMA and had a significantly lower risk of recall. However, both pathology (28%) and cardiovascular (3%) had a higher percentage of devices with PMA, without increased risk of recall. Ghobadi et al7 reported that 71% of high-risk recalls were for devices with 510(k) clearance, a number slightly lower than seen in this study, but they only examined recalled devices and did not count multiple recalls for the same device. To our knowledge, there is little existing literature analyzing devices with multiple recalls; however, the present study found that nearly one-third of recalled devices for both PMA and 510(k) clearance were recalled multiple times, with several devices in each group noted to have multiple class I recalls. This finding suggests that certain devices continue to raise safety concerns even after an initial recall, especially in those with multiple class I recalls. Caution, however, is urged in overinterpreting these results because the reasons for the subsequent recalls is unknown and beyond the scope of this investigation.Last, this study found 5362 recalls in the 12-year analysis period, with 97% for devices with 510(k) clearance and 3% for those with PMA. Zuckerman et al32 however, found no significant increase in recalls associated with the passage of these amendments from 2007 to 2016. Next, recalls are highly dependent on adverse event reporting, particularly through the Manufacturer and User Facility Device Experience (MAUDE) database, which the FDA reports potentially includes inaccurate and incomplete data.33 Adverse events are also more likely to be identified for devices that are used more frequently, a confounder not readily adjusted for and not done in previous studies. Furthermore, a device can raise major safety concerns without being recalled. Two particularly publicized examples are the Bayer Essure device for permanent sterilization34 and laparoscopic morcellators,35 which have been linked to iatrogenic dissemination of potentially cancerous cells within the peritoneum. In neither case were recalls formally issued, but the FDA did publish multiple public updates and guidance documents. The Essure device was voluntarily withdrawn from the market by the manufacturer without a formal recall. If other devices were voluntarily withdrawn, they were not accounted for in this study or in most previous studies. This outcome is owing to inconsistent reporting in the FDA database and the fact that the FDA considers withdrawals to “involve a minor violation that would not be subject to legal action by the FDA.”31 Finally, recall is a surrogate end point, not necessarily capturing the patients’ level of pain or the extent of damage to public health. As an example, the Depuy ASR (Articular Surface Replacement) metal-on-metal hip implant recall, despite being a class II recall, has left a significantly larger impact on public health than the class I recall of the Echelon Flex Endopath Stapler (510(k) number: K081146), which cleared the 510(k) pathway the same year.36 These issues create an inability to capture all device-related safety concerns and are associated with a probable underestimation of the real problem, although we believe these results reflect the most accurate to date.There are several important limitations to the study. First, several FDA policy changes were passed during the study period, such as the Medical Device User Fee Amendments, which increased funds that often support factory inspections and other surveillance activities, possibly leading to the discovery of more recalls. Ghobadi et al,38 Recently implemented by the FDA is the Global Unique Device Identification Database, in which the FDA tracks devices with a unique device identifier.39 This identifier will eventually be linked to electronic health records and possibly with certain high-quality registries. We also agree with calls to increase the quality of evidence used in the pivotal trials performed for devices with PMA.29 Third, many unanswered questions remain regarding device safety, including the variability in recalls between specialties and individual devices, and further research is indicated to help safeguard the public health.In this cohort study, despite requiring clinical trials, the high-risk devices approved through PMA were associated with a substantially greater risk of recall than previously reported. Second, devices with 510(k) clearance accounted for most recalls, and the 13% risk of recall at 9 years likely underestimates safety problems, as previously noted. Although not specifically addressed in this study, we recognize the importance of balancing the need to ensure safety and effectiveness with bringing innovative treatments to patients as quickly as possible, and we join with multiple other authors in calling for increased postmarketing surveillance strategies to supplement the MAUDE database and Medwatch program. Health professionals can, and should, register device safety concerns with the FDA through Medwatch, although participation is voluntary.