Poor responder patients remain a challenge in assisted reproductive technologies. The “short agonist stop” (SAS) stimulation protocol uses a double stimulation (flare up effect with the gonadotropin-releasing hormone (GnRH) agonist (GnRH-a) then gonadotropins) associated with a less strenuous blockage (discontinuation of GnRH-a) to favor follicular recruitment in order to obtain a better ovarian response. This study aims to compare the number of oocytes obtained after a SAS stimulation protocol with those obtained after the previous stimulation protocol, in the same women, with poor ovarian response (POR) diagnosed according to the POSEIDON criteria.This therapeutic observational retrospective cohort from 2018 to 2022, with a case-control evaluation compared with the same patients’ previous performance, included women with POR undergoing IVF with SAS stimulation protocol. The primary outcome was the number of total oocytes recovered and secondary outcomes were the numbers of mature oocytes, total embryos observed at day 2 and usable cleaved embryos and blastocysts (day 5/6).63 patients with SAS and previous cycles were included. In the SAS group, the mean number of oocytes was significantly higher: 7.3 vs 5.7, p=0.018 in comparison with the previous attempt. So was the number of mature oocytes  and the total mean number of embryos obtained at day 2 . The SAS stimulation generated 84 usable embryos: 57 cleaved embryos and 27 blastocysts. The mean number of usable embryos was similar in both groups . In total, out of 63 patients, after the SAS protocol, and subsequent embryo transfers , 9 patients had ongoing pregnancies and no miscarriage occurred. The cumulative ongoing pregnancy rate (cOPR) after the SAS protocol was 14.3% (9/63) per oocyte pick-up and 16.7% (9/54) per transfer.SAS stimulation is a short and original protocol strengthening the therapeutic arsenal of poor responders, that may offer promising results for those patients with low prognosis and previous failed IVF. Results must be confirmed with a randomized controlled trial. Poor responder patients remain a challenge in assisted reproductive technologies (ART). The definition of this heterogeneous group has long been debated. In 2011, the European Society of Human Reproduction and Embryology (ESHRE) reached a consensus on the definition of ‘poor ovarian response’ (POR) to ovarian stimulation called the Bologna criteria: at least two out of three features must be present: advanced maternal age (≥40 years), a previous POR (≤3 oocytes) with a conventional stimulation protocol or an abnormal ovarian reserve test  <7 follicles or anti-Müllerian hormone (AMH) <1.1 ng/ml)  associated with a less strenuous blockage (discontinuation of GnRH-a) to favor follicular recruitment in order to obtain a better ovarian response and produce more oocytes and embryos, including more usable embryos, increasing chances of ongoing pregnancies (OP) in these low prognosis patients , 15. BasThe SAS protocol as presented in this study is not mentioned in recent literature about stimulation protocols and management of poor responders in ART , 20, norTherefore, this pilot study was performed to evaluate if in poor responders’ patients, the SAS stimulation protocol allows for a better number of oocytes (primary outcome), mature oocytes, total embryos at D2 and usable embryos (secondary outcomes) in comparison with the last previous IVF attempt within the same patients, in order to obtain more ongoing pregnancies in those patients with numerous previous failed IVF attempts.st, 2018, and January 1st, 2022  were included.This was an observational, retrospective, single-center study with case-control evaluation compared with the same patients’ previous performance in their last IVF attempt. All patients enrolled in an IVF protocol with the “short agonist stop” (SAS) stimulation protocol at Montpellier University Hospital between January 1The main objective of the study was to compare, in poor responders, the number of oocytes obtained after the SAS stimulation protocol versus previous classical stimulation of their last IVF attempt, each patient being their own control.The main outcome measure was the total number of oocytes obtained in poor responder patients after the SAS stimulation protocol.Secondary outcome measures were the total number of mature oocytes, embryos at day 2, usable embryos , cancellation and freeze all rates, and the outcome of IVF attempt: no pregnancy, biochemical pregnancy, miscarriage, ectopic pregnancy, ongoing pregnancy  and live birth (if available), including cumulative outcomes.Patients included in the study were ≥ 18 and < 43 years old. Preimplantation genetic testing and fertility preservation cycles were excluded. Non-compliance to the SAS protocol (modification of duration of GnRH-a) and lacking data in previous cycle were exclusion criteria. Only the first cycle with SAS was included compared with the previous stimulation cycle. Women with defined POR (low prognosis patients) were included, according to POSEIDON stratification.Registered patient data included age, AMH rate, AFC, body mass index (BMI), POSEIDON’s group and the rank of the attempt. Measured parameters included type of protocol, days of stimulation, dose and type of gonadotropin, total dose used of gonadotropin, and all outcome measures as detailed above.Patients were informed of the investigation and gave their consent before participation. This study was approved by the local Ethics Committee (IRB registration number 202201068).Enrolled patients were treated in two consecutive cycles. The first attempt was achieved with a standard protocol for POR: antagonist protocol, long or short agonist protocol and mild stimulation, summarized in ®, 250 mg, Laboratoire Merck, France) when the leading follicles  reached a mean diameter of 17 mm and oocyte retrieval was performed 35 hours after. If less than 3 follicles were recruited, a conversion to ovulation induction or intra uterine insemination was performed, if possible (at least one patent tube and adequate semen parameters). Luteal phase was supported similarly by daily vaginal administration of 600 mg of micronized progesterone , until the pregnancy test.Ovulation triggering was performed with recombinant hCG  were treated in the subsequent cycle with the SAS protocol. Clinical and laboratory aspects of treatment were mainly done in a similar fashion in both cycles if the first cycle was done in Montpellier University Hospital, each patient acting as her own control.®, Laboratoire Sanofi-Aventis, France), starting in the midluteal phase (D20) of the preceding cycle, was prescribed. After an ultrasound to confirm ovarian quiescence and a thin endometrium, associated with low P serum on day 2 of the cycle, 0.1 mg triptorelin  was initiated daily, for 7 days, then stopped. Controlled ovarian stimulation (COS) was initiated 2 days after the beginning of the GnRH-a, on day 4, with FSH or hMG at 300 IU as a starting dose. After 7 days without agonist, GnRH antagonist, ganirelix 0.25 mg  was used daily until triggering. Triggering, oocyte retrieval and luteal phase support were all performed in a similar fashion to the previous IVF attempt.For the SAS protocol Figure 1Conventional IVF (cIVF) or intra cytoplasmic sperm injection (ICSI) technique were used as appropriate. IVF procedure was performed in our unit as previously described . At D3, Fresh embryo transfers were performed either at the cleavage (D2-D3) or blastocyst stage (D5). Early blastocysts (grade 1 or 2) at D5 were kept in culture until D6 and cryopreserved if considered usable at that point. The surplus embryos (D2-D3 or D5-D6) that were considered usable according to morphologic criteria were cryopreserved for subsequent transfers. The embryo transfer strategy was determined by a multidisciplinary team. Embryos were cryopreserved by vitrification and thawed following the manufacturer’s recommendations . A maximum of two embryos were replaced.All usable embryos were frozen  for subsequent frozen ET cycles if the circumstances were unsuitable for fresh ET, for instance in case of elevated P level, inadequate uterine cavity, prolonged ovarian stimulation (> 13 days) or accumulation of vitrified embryos for later transfer (desynchronization). FET (frozen embryo transfer) cycles were performed with natural cycle, hormonal replacement therapy or stimulated cycle regarding the ovulatory status.Pregnancy was assessed by serum hCG assay after 15 days from oocyte retrieval. A biochemical pregnancy is characterized by the absence of an identifiable pregnancy on ultrasound examination despite a positive blood hCG pregnancy test (<100 IU/L). Clinical pregnancy was confirmed if a fetal heartbeat could be observed by transvaginal ultrasound. An ongoing pregnancy (OP) is defined as a pregnancy with a detectable heartbeat at 12 weeks of gestation or beyond. Live birth is defined as the birth of at least one living child, irrespective of the duration of gestation. Cumulative OP rate (cOPR) includes the outcomes from all fresh and frozen embryo transfers following an episode of ovarian stimulation.Quantitative variables were reported with the usual statistics: mean and standard deviation, median and range, and were compared between the previous IVF simulation and SAS protocols using sign rank test (paired tests were performed because each subject is their own control). Categorical variables were reported with the number of observations (N) and the frequency (%) of each modality and compared using a McNemar test (paired tests were performed because each subject is their own control). All tests were two-sided and p-values < 0.05 were considered as statistically significant. Statistical analyses were performed with STATA 15.0 .Out of 100 patients, a total of 63 patients who underwent IVF with the SAS protocol consecutively to a previous stimulation cycle were included in the final analysis Figure 3Almost half of the patients belong to POSEIDON’s group 4 (47.6%). The previous stimulation protocol occurred almost 8 months  before SAS and was in most cases an antagonist protocol (76.2%). HMG were mostly used compared to rFSH in both protocols: 66.7% (previous) and 79.4% (SAS).The cancellation rate (19.1%) was the same in both groups. It was generally due to insufficient ovarian response (less than 3 follicles recruited) (66.7% and 75.0% in the previous and SAS cycle respectively). In the SAS group, there was no cancellation due to premature ovulation. In both groups, 51 patients proceeded to oocyte retrieval . In both groups, ICSI was mostly performed (72.55 and 88.24%) Table 2.In the SAS group, 7.3 oocytes were retrieved versus 5.7 in the previous attempt, which was significantly higher (p=0.018). The mean number of metaphase II oocytes was also significantly higher: 5.8 versus 4.1(p=0.032) as well as the total mean number of embryos at day 2: 4.1 versus 2.7 (p=0.016). The freeze all rate was significantly higher in the SAS group: 47.1% (24/51) versus 13.7% (p=<0.001), mostly due to excessive length of stimulation (70.8%). There was no difference between groups in cycles without usable embryos  Table 2.There was no statistically significant difference in the embryo stage for those transferred (p=0.507). The number of cumulative ET in the SAS group was higher: 54 vs 42, but with no statistical difference (p=0.124). In the previous cycle group, there were more fresh ET (40.7 vs 78.6%) than in the SAS group, which had more frozen ET (37.0 vs 14.3%). Both groups had similar ET with cleavage embryos (68.5 vs 73.8%) but in the SAS group, there were more ET with blastocysts: 24.1 vs 16.7%. The mean number of usable embryos was higher in the SAS group than in the previous IVF: 1.6 vs 1.3, but with no statistical significance (p=0.178) Table 3.In the previous attempt group, 5 biochemical pregnancies and 6 miscarriages occurred. There was no ongoing pregnancy.rd trimester). OP occurred in patients belonging to POSEIDON’s group 3 (n=5), group 4 (n=2) and group 1 (n=2).No miscarriage occurred after the SAS protocol. The cOPR in the SAS group was 12.0% per cycle, 14.3% per ovarian puncture and 16.7% per transfer Table 4.This retrospective study has documented that SAS stimulation is a short and original protocol strengthening the therapeutic arsenal of poor responders, which may offer promising results for those patients with low prognosis and a record of failed IVF. This protocol resulted in a significantly higher number of oocytes, mature oocytes, and embryos obtained and a non-significantly higher number of usable embryos, in comparison with their previous IVF cycle.In Loutradis and Badawy et al.’s review, for poor responders, GnRH agonist flare up and long agonist protocols did not seem to be as advantageous as a reduction of GnRH-a doses, “stop” protocols, or microdose GnRH-a flare regimens. These regimens all appeared to improve outcomes, although the benefit of one approach over another has not been convincingly established , 25, witThe use of a GnRH-a during COS in long protocols may lead to poor ovarian response due to intense endogenous FSH suppression  and the In the SAS group, significantly higher oocytes and mature oocytes were retrieved than in the previous attempt. Cumulative LBR is considered to be the most important outcome in IVF and it is in direct association to oocyte yield following COS . In SunkOn the one hand, in a small cohort , poor reWe found that the mean number of usable embryos were higher in the SAS group with no statistical significance. The number of cumulative ET in the SAS group was higher: 54 vs 42, but with no statistical difference (p=0.124). Twelve surplus embryos are waiting for ET (mainly because of an ongoing pregnancy), so the number of cumulative ET would probably be significant if all embryos were transferred, with potentially more pregnancies. Schachter et al. also found significantly more cleaving embryos with improved morphology after discontinued GnRH-a protocol in comparison with long agonist protocol .The freeze all rate was significantly higher in the SAS group mostly due to prolonged stimulation, indication based on the fact that prolonged stimulation is associated with decreased ART success because of impaired endometrium for implantation (except for PCOS) –41. HoweThe cOPR in the SAS group was 14.3% per ovarian puncture and 16.7% per transfer. One study suggests that the LBR in POR patients ranges from less than 1 to 10% per cycle . Other dOtherwise, the miscarriage rate (MR) in the SAS group was particularly low, probably because of the small size of our population and of the selection of our population with previous failed IVF cycle . There is no reason to believe that the SAS stimulation could reduce miscarriage by enhancing the ploidy rate, because recent studies show that ovarian stimulation does not impact the risk of aneuploidy , 46. OthSome limits to our study must be pointed out. The main limitation is its retrospective nature and the modest sample size. Due to the design of the study, the outcome of IVF (OPR or LBR) cannot be chosen as the main outcome measure because we proceeded to another IVF attempt precisely because the previous IVF failed. The heterogeneity of the group of previous attempts is another limit. Different protocols were used with different pretreatments: estrogens priming (76.2%), daily agonist desensitization (17.46%) or no pretreatment (6.34%). IVF was sometimes performed in another center (n=16) with lacking data (like duration of the stimulation) and with different laboratory techniques, but this rather reflects the reality of poor responders’ care and patients remain the same. In addition, the cancellation rate affecting different patients (n=8) in both groups is a limit, as our main outcome was the number of oocytes retrieved, occurring after oocyte pick-up. Furthermore, the comparability of both groups is limited: the time between both attempts could be associated with some changes in variables such as BMI, obviously age (but in favor of previous attempt), AMH, sperm etc. Yet, the delay in between is sufficiently short  that no major differences are to be expected, as found in Romanski et al.’s study, showing that the delay in IVF treatment up to 180 days does not affect pregnancy outcomes in women with diminished ovarian reserve . There wThe strength of this study lies in the comparison of the same patients’ performance in consecutive cycles, which presents the advantage of controlling for differences that might be expressed in randomized patient groups. Patients acting as their own controls  make the study less biased. The stimulation protocol is original and has only been used in one published study . The benThe results of this pilot study need to be confirmed with a prospective trial to assess the genuine usefulness of this protocol and to decipher which type of poor responders would benefit the most from SAS stimulation.In conclusion, the SAS stimulation protocol may offer promising results (more mature oocytes and embryos) for poor responders with low prognosis and previous failed IVF. Those results must be confirmed with a large prospective study such as a RCT evaluating live birth rate after SAS protocol versus standard protocol. The SAS original protocol might strengthen the therapeutic arsenal of poor responders and enable a more tailored management.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.CM is the main author. CM collected all the data, wrote the manuscript and designed the figures, working under the direction of TA. The methodology was elaborated in association with TA and CMo. CMo performed the statistical analysis and made the tables. NR, CB, SH, and SB were proofreaders and enhanced the manuscript with their corrections. 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.


Dermogenys collettei), which invest in sexual ornaments and routinely engage in male–male contests and sperm competition. Critically, we examined responses in males when resources were restricted during development and after reaching sexual maturity. Both pre- and post-copulatory traits are resource-dependent in male halfbeaks. Body size, beak size, courtship behavior, and testes size were reduced by diet restriction, while, unexpectedly, the restricted-diet group had a larger area of red color on the beak and fins after diet treatment. These patterns were generally consistent when resources were restricted during development and after reaching sexual maturity. The study reinforces the role of resource acquisition in maintaining variation among sexual traits.Exaggerated and conspicuous sexually selected traits are often costly to produce and maintain. Costly traits are expected to show resource-dependent expression, since limited resources prevent animals from investing maximally in multiple traits simultaneously. However, there may be critical periods during an individual’s life where the expression of traits is altered if resources are limited. Moreover, costly sexual traits may arise from sexual selection acting both before (pre-copulatory) and after mating (post-copulatory). Gaining a robust understanding of resource-dependent trait expression therefore requires an approach that examines both episodes of sexual selection after resource limitation during different times in an individual’s life. Yet few studies have taken such an approach. Here, we examine how resource restriction influences a set of pre- and post-copulatory traits in male pygmy halfbeaks ( Hide Does going on a diet make animals worse at reproducing? Evolutionary theory suggests that reproductive success often depends on how good animal are in finding food and converting this energy into finding and attracting mates. We tested this idea in a small freshwater fish and show that males with access to less food are worse at courting females, have shorter jaws, and produce fewer sperm. Sexual selection is expected to promote the evolution of traits that increase success in intra-sexual competition and inter-sexual mate choice . Such seGnatocerus cornutus, A powerful method to examine the resource dependence of sexual traits is by experimentally manipulating resource availability. Experimentally restricting available resources reduces or alters sexual behaviors and the phenotypic expression of a range of sexual traits in numerous animal groups . For exaSexual selection can also favor the evolution of exaggerated post-copulatory traits linked with fertilization success during sperm competition, such as sperm number and quality , 2020. AWhile costly sexual traits are generally expected to exhibit resource-dependence, such effects may fundamentally depend on when resources are limited during an individual’s lifetime. Specifically, responses to resource restriction may depend on an individual’s developmental stage, as limiting resource during development versus after reaching sexual maturity may influence trait expression differently e.g., . MacartnDermogenys collettei). Pygmy halfbeaks are small, viviparous, internally fertilizing, and promiscuous fish from Southeast Asia. Halfbeaks are characterized by an elongated lower jaw , and large post-copulatory investment makes the halfbeak an ideal model to examine resource dependence of a range of sexually selected traits. We capitalize on these attributes to contrast patterns of resource dependence across sexual traits after resource restriction during either juvenile development or after reaching sexual maturity in male halfbeaks. We then assess whether resource restriction in males influences the outcome of male–male competitive interactions and female mate preferences in halfbeaks. We predict that overall, 1) sexual traits are costly to produce and maintain and will therefore show reduced expression after resource restriction, 2) resource-dependent trait expression will be stronger when resource restriction occurs during development than when applied after males are sexually mature, in particular for structural traits (such as beak length), 3) females will prefer males with more exaggerated expression of resource-dependent sexual traits, and 4) males on resource-restricted treatments will be competitively inferior during male–male competition.Experimental fish were generated from F1 and F2 descendants of wild-caught halfbeaks collected from Sungai Tebrau, Malaysia, that were bred in an aquarium facility at Stockholm University, Sweden. The adult fish, which live for up to ~3 years in captivity , used to produce the experimental subjects were kept in mixed-sex groups in 160 L aquaria containing ~2 cm of gravel, aeration, and live and artificial plants. Fish were maintained on a 12:12 h light:dark cycle at ~26 °C. Offspring were collected either from mixed-sex groups or individually housed gravid females  and then used in one of the two diet manipulation experiments described below. The two experiments were performed consecutively over the course of 6 months in 2019 and 8 months in 2020, respectively. Experiments were approved by Stockholm Animal Research Ethical Board (permit number 2393-2018 and 3967-2020).adult diet experiment). Offspring were reared until sexual maturity under standard laboratory conditions (see above). After birth, up to five offspring (~3–5 mm in length) were housed together in 4 L tanks for up to 2 months, during which they are fed exclusively live Artemia salina nauplii (henceforth referred to as artemia) twice per day. 20–30 fry from different 4 L tanks that were born within the same calendar month were then moved to 72 L tanks until the onset of sexual maturity. During this period of development, juveniles were fed ad libitum with a mixture of ground flake food and freeze-dried artemia twice per day, 6 days per week. In addition, previously frozen and thawed Drosophila melanogaster and live artemia were fed to fish once and 3–4 times per week, respectively. At the onset of sexual maturity (~2 months of age), males were identified by the developing of the modified anal fin (the andropodium) used for copulation  were taken from male-only tanks at age ~3–4.5 months and placed in individual 4 L tanks (25 × 16 × 12 cm) containing ~2 cm of gravel, four floating plastic plants, 2–3 snails, and constant aeration. A floating circular feeding arena (a hollow plastic tube) was placed in the center of the tank. The feeding arena was used to ensure that fish could easily find and access the food. Fish were allocated to one of two experimental diet treatments: a high or restricted quantity diet. There was no difference in body size between groups prior to diet treatment . Of the original sample size of 70 males, 4 died during diet treatment (2 from high diet and 2 from restricted diet treatment), reducing the final sample size to 66 males (33 in each treatment). In the adult high-diet treatment (n = 33), each week males were fed six fruit flies (D. melanogaster) per day  for 5 days and 3 flies per day for 2 days . In the adult restricted-diet treatment (n = 33), each week males were fed one fruit fly per day for 5 days and 2 flies per day for 2 days . We used only fruit flies to manipulate adult diets because they present a simple way to control food quantity. Males were maintained on these diet treatments for 28–31 days. Note that assays were initiated around day 21 of the diet treatment and continued until termination at day 28–31. In all tanks, approximately 10% of the water was changed three times per week, using a flow through system to minimize disturbance. The adult diet experiment was performed in 4 blocks .To experimentally manipulate adult diets, sexually mature males (Assays (described below) were performed in the following order, each separated by 1–3 days: female mate choice assay, courtship assay, competition assay, morphological and post-copulatory measurements .developmental diet experiment). New-born fish  were collected from stock tanks and placed in 4 L experimental tanks. For the first 28 days of the experiment, juveniles were kept in groups of 2–4 individuals and fed an ad libitum diet of live artemia twice per day. This initial ad libitum diet stage was done to minimize juvenile mortality, which is greatest during the first month after birth. After these initial 28 days, juveniles were placed in individual 4 L tanks containing 2 cm of gravel, plastic plants, 2–3 snails, and constant aeration. Juveniles do not eat D. melanogaster and were therefore maintained on a diet of live artemia. Juveniles were allocated to either a high or restricted-diet treatment. In the developmental high-diet treatment, fish were fed 3 ml of the solution obtained from 6 ml artemia cysts hatched in 1200 ml water solution twice per day for 6 days each week (12 × 3 ml feedings per week). Fish in the restricted-diet treatment were fed increasing amounts of food as they grew after the initial 28 days of ad libitum feeding to account for increases in fish size throughout the experiment. The same concentration of artemia was used throughout the experiment. During the first, second, and third month of the diet treatment, juveniles in the restricted-diet treatment were fed 3 ml of the artemia solution 1) once per day, 3 days per week (3 × 3 ml feedings per week), 2) once per day, 5 days per week (5 × 3 ml feedings per week), and 3) twice per day for 1 day per week and once per day for 5 days per week (7 × 3 ml feedings per week), respectively. Fish were maintained on these diet treatments for a total of 3–3.5 months, which included the last 1.5 week during which assays were performed.In this experiment, halfbeaks were reared on different experimental diets treatments until reaching sexual maturity . At 4–4.5 months of age, the assays were initiated. Note that since all assays take approximately 10 days to complete per block, diet treatments continued during this time. The order of the assays was the same as in the adult diet experiment (see above) with the exception that a lateral picture (see below) was captured the day before female mate choice in order to produce the size-matched pairs of males.Of the initial 111 juveniles that entered the experiment, 10 died over the course of the experiment , reducing the sample size to 101 fish . The experiment was performed in three blocks, where fry born within 2 weeks were pooled into a single block and assayed together. Each of the blocks contained between 32 and 36 juveniles, with diet treatments equalized within each block. When focusing only on males, this translated into 18–25 males per block. The final sample size in the developmental diet experiment was 50 males .Body size and external morphology were quantified for males in both experiments using photographs. The lateral side of each male was photographed using a digital camera (Canon 800D with macro lens EF-S 60 mm) inside a photo chamber (30 × 20 × 20 cm) under standard light conditions. Each image included a scale bar. From these images, body length was measured in ImageJ v1.52i;  from theTo examine coloration on the fins and beak, we used a principal component analysis (PCA) to reduce the dimensionality of the data, thereby reducing the number of statistical tests (and the associated risk of Type-II errors) and account for potential collinearity among variables. The total amount of red, yellow, and black coloration on the fins and red and black coloration on the beak were entered into a PCA. The PCA returned 2 PCs (henceforth coloration PC1 and PC2) with standard deviations ≥ 1, which together accounted for 56% of the total variance and were used for further analysis . LoadingK = (body mass/length3) * 100), a common proxy of condition in fish . This procedure was repeated for two independent mass measurements per individual and an average of the two measurements was used for analysis. Body length and mass were measured at the start and end of the diet treatments in the adult diet experiment, while measures were only taken at the end of the diet treatments in the developmental diet experiment. We estimate body condition using Fulton’s condition factor  where the male twists his body to attempt to insert the andropodium into the females genital pore . Fish were fed ~2 h prior to all courtship assays. Trials for all courtship assays were performed and analyzed blind to the treatment group by giving the males a unique identifier code.Male courtship behaviors were recorded using a free-swimming assay where one focal male could interact with one female. Individual males from each of the adult and juvenile diet treatment groups were added to an experimental tank (dimensions 40 × 24 × 30 cm) filled to a water depth of 15 cm, containing two plastic plants and ~1 cm of gravel. A sexually mature  virgin female randomly chosen from a female-only stock tank was then added to the experimental tank. The male and female were separated by a transparent plastic divider and left undisturbed for 1 h, allowing the fish to habituate to the experimental tanks. After the habituation period, the transparent divider was lifted using a pulley system, allowing the male and female to interact. During a 20 min observation period, we recorded a number of behaviors. As discrete behaviors, we recorded the number of male total number of sexual behaviors index. Similarly, all behaviors measured as durations  were summed together to generate a total duration of sexual behaviors index. After summing the behaviors, these indices were divided by the amount of time that the focal male was aware of the female, which was obtained by subtracting the time from the start of the trial until the male and female first interacted from the total time of the trail. Courtship trials were aborted if the male or female showed signs of stress  or if either fish acted aggressively towards the other . Thus, the final sample size for the courtship assays was 106 trials .We summed the behaviors measured as counts (circling and mating) to generate a Competition behaviors were video-recorded in a free-swimming assay where one male from the high-diet treatment and one from the restricted-diet treatment were allowed to interact. To examine the effect of resource restriction on competitive interactions removing body size-mediated effects, males in each trial were size matched prior to the assay with a maximum difference of 3 mm in length allowed between the males in the pair. On average, the difference in length within the pair was 0.44 ± 0.08 mm (mean ± SE) in the adult diet experiment and 1.43 ± 0.16 mm in the developmental diet experiment. In two blocks of the developmental diet experiment, there were two more fish in the juvenile restricted-diet treatment than in the juvenile high-diet treatment. Therefore, these four fish created pairs with two males of the same treatment. These pairs were treated the same as regular pairs , in order for all fish to have the same experience for subsequent assays. These two recordings were not included in the analysis (see below).performed by the focal fish and directed at the rival fish or the number and duration of behaviors that were experienced by the focal fish . Lunges and displacements were considered aggressive behaviors. In total, 58 trials were recorded . Scoring of behaviors stopped before the end of the recording  if the observer lost track of the IDs of the experimental individuals (n = 10).The competition assays were recorded from above with a camera (Point Grey Grasshopper 3 4.1 megapixel camera with Fujinon CF25HA-1 lens) placed 1.5 m directly above a circular experimental tank . Each male in a pair was first placed in a separate opaque cylinder (15 cm in diameter) inside the experimental tank and allowed to acclimate for 15 min without physical or visual contact with the other male. After the acclimation period, the recording was initiated, and the cylinders were lifted by hand. The recording proceeded for 20 min. The video recordings were subsequently scored blind by one experimenter (E.F.I.). In each trial, a randomly chosen focal individual was selected and their behaviors were recorded by keeping track of the focal fish frame by frame . We scored the number of displacements , the number of lunges , and the duration of swimming behind . All behaviors were scored from the perspective of the focal fish, recording the number and duration of behavior experienced by the focal individual to performed by the rival, in order to have a comparable value for the males of the two treatments. Therefore, the data was analyzed per individual, rather than per recording. Since each recording is represented two times in the data, we also control for recording ID in the analysis. The behaviors measured as counts (displacements and lunges) were summed per individual. Count behaviors and the duration behavior were divided by the total trial time. Trials had to be a minimum duration of 10 min of interactions to be included in the analysis, thus excluding four shorter trials. Three recordings were excluded due to technical errors while recording. Two recordings were excluded since the males in the pairs were from the same diet treatment (see above), leading to a final sample size of 100 individuals .For analysis of competitive behaviors, we converted the behaviors A dichotomous choice assay was used to assess whether male resource-dependent traits influence the outcome of female mate preferences. For both the adult and developmental diet experiments, one female and two males (one from each diet treatment) were placed in an experimental tank (45 × 25 × 20 cm), consisting of one main chamber (45 × 15 × 20 cm), and 3 adjacent, isolated stimulus chambers  or a webcam  positioned 30 cm above the tank. The duration of time a female spent in the left or right association zones (an area of 5 × 15 cm in front of each stimulus chamber) was used as a measure of female preference for the male in that chamber. Association time is a commonly used proxy of mating preference, which is predictive of preference during real mating interactions . To quann = 7), or visited him late into the video, resulting in less than 10 min of informed choice (n = 3). Two recordings from the developmental diet experiment where both of the two males in the pair were of the restricted diet treatment were removed (n = 2). Additionally, trials were excluded if the female or one or both males behaved in a stressed manner (n = 4). The final sample size consisted of 39 trials .In total, 55 trials were conducted. In a few cases, the female never visited the second male , washed in deionized water and placed on a glass slide containing ~1 ml of a 9% saline (NaCl) solution, and viewed under a dissection microscope . Male halfbeaks produce sperm bundles called spermatozeugmata . Sperm bn = 30; restricted n = 32; juvenile diet manipulation, high n = 23, restricted n = 23). Only velocity samples with a minimum of 20 sperm cells measured were used, and the threshold for defining static cells was predetermined at 25 µm/s for VCL. Applying these exclusion criteria reduced the final sample size to 93 males . From these males, sperm velocity was measured for a mean (±SE) of 102.76 ± 12.17 and 183.59 ± 15.52 sperm cells per male in the adult and juvenile diet manipulation experiments, respectively. We focus on three commonly used, colinear sperm velocity metrics obtained from CASA, including the average path velocity (VAP), straight-line velocity (VSL), and curvilinear velocity (VCL). These three sperm motility measures were reduced using a PCA. The PCA returned one PC with standard deviation ≥ 1, which accounted for more than 96% of the total variance and was used for further analysis . Since they are measured from different subsamples, sample size may differ between traits (see below). To assess sperm velocity, 3 µl of the sperm/saline solution was transferred to two wells of a 12-well multitest slide  coated with a 1% polyvinyl alcohol (PVA) solution and covered in a previously coated PVA cover glass to prevent cells sticking to the glass . Sperm sanalysis . Sperm vanalysis .n = 30; restricted n = 29; developmental diet experiment, high n = 23, restricted n = 24).Sperm viability, quantified as the proportion of live sperm cells in each ejaculate, was measured using a live/dead cell viability kit . A 14 µl subsample from the sperm/saline/HBSS solution was transferred to a new Eppendorf tube and 1.6 µl of propidium iodide and 0.5 µl of acridine orange were added. After adding the dyes, 14 µl (2 samples of 7 µl each) of stained sperm solution was transferred to a microscopic slide and left for 1–2 min in darkness, after which it was placed under a fluorescent microscope, and images were captured . Viable sperm with an intact outer membrane are labelled green by membrane-non-permeable dye (acridine orange) while dead or inviable cells with disrupted membranes are labelled red with membrane-permeable stain (propidium iodide). Sperm viability was then calculated based on a mean (±SE) of 244.6 ± 34.7 cells (range 25–1383) in the adult diet-manipulation experiment and 311.3 ± 4.2 cells (range 74–1046) in the developmental diet experiment. The total number of cells that were counted in the samples is accounted for in the analysis (see below). Of a total of 116 males from both experiments, the viability subsamples of 10 males had insufficient sperm, reducing the sample size for sperm viability to 106 males .For sperm morphology measurements, images of sperm cells were captured from each male’s ejaculate . Pictures were analyzed in ImageJ to measure length of head, midpiece, and flagellum . The three measures of sperm morphology were condensed using a PCA, which returned one PC (henceforth sperm morphology PC1) with standard deviation ≥ 1, which accounted for 42% of the total variance and were used for further analysis . Sperm mn = 29; restricted n = 30; developmental diet experiment, high n = 20, restricted n = 26).After sperm assays, the male was dissected and the testes were removed under a dissection microscope  and weighed . We removed one male (adult high-diet treatment) from the analyses as his testes were not properly developed which reduced the sample size to 115 males  and when the diet experiments were applied , all models initially included diet treatment, experiment, and their interaction as fixed factors (unless otherwise stated).First, we validated that male body condition was influenced by the diet treatment. To do this, we use a linear model with male condition (Fulton’s K) as the response variable and the diet treatment, the experiment , and their interaction were entered as fixed factors. In the adult diet experiment, data on male condition were also available at the start of the diet treatment. Therefore, we compared adult condition (response variable) before and after the diet treatment in a linear mixed model with diet treatment, timepoint (at the beginning or end of the diet treatment), and their interaction, as fixed factors, and male ID as random factor.Next, we examined if diet treatment and timing of diet manipulation  influenced the expression of putative pre-copulatory sexually selected traits (beak length and coloration area) as well as male courtship  and competitive behaviors . These (apart from competition behavior) were analyzed using linear models that included the trait as a response variable and diet treatment, experiment, and their interaction as fixed factors. The interaction was removed if non-significant. Competition behavior was analyzed using a linear mixed model to additionally include recording ID as random factor since the analysis is done per individual and not per recording. Body length was added as a covariate to models examining beak length to account for allometric effects. In models with coloration (coloration PC1 and PC2), body length and beak length were added as covariates. In models assessing male courtship behaviors, the total number of courtship behaviors were log10-transformed and the total duration of courtship behaviors was square-root transformed to improve the fit to a normal distribution.Next, we examined whether females preferentially associate with the male of one treatment over the other using a strength of preference (SOP) score for the left male (arbitrarily defined without regard to diet treatment) as the time a female spends in the association zone of the left male divided by the total amount of time the female spend in the left and right association zones. SOP values range from 0  to 1 . We then fit a linear model with SOP  as the response variable, with diet treatment and experiment as fixed factors. To examine if male morphological traits influenced female preferences, we first calculate the difference in morphological trait values between the left and right male regarding body length, beak length, red coloration on the fins, red on beak, black on beak, yellow coloration on the fins, and black coloration on the fins. A linear model was fitted with SOP for the left male as the response variable and the differences in trait values together with experiment as fixed factors. The results did not change when we fitted each trait in separate models.Finally, we examined the effect of diet restriction on sperm velocity, sperm viability, sperm morphology, and testes mass. For sperm viability, we calculated the proportion of live sperm in the samples from each male. Post-copulatory traits were assessed using linear models that included the trait as response variable and diet treatment, experiment, and their interaction, as fixed factors. Body length was added as a covariate to models examining testes mass to account for allometric effects. The model with sperm viability was weighted by the total number of cells counted .lm function from the stats package for linear models. The lmer and glmer functions from lme4 package . Across all trials, 3.82 ± 0.26 (mean ± SE) displacements occurred per trial. Lunges occurred in 50% of trials  and 4.11 ± 0.35 (mean ± SE) lunges occurred per trial. Beak-locking was less common and occurred in only 23% of trials . However, there was no difference in the number of competitive behaviors performed by males in either diet treatment or between the experiments . SimilarF = 0.1020, df = 1, p = 0.32), and this was consistent between the two experiments . Female preference was also not influenced by the difference in male body length , beak length , red coloration on the fins , red on beak , black on beak , yellow coloration on the fins , or black coloration on the fins , and this did not differ between experiments .Male diet treatment did not influence who the females spent more time with , experimental treatment , and their interaction  in the full dataset. However, these sperm viability effects were driven by three outliers. When these outliers were removed from the model, sperm viability was not influenced by diet treatment  conditions for assaying). Specifically, the duration of courtship behaviors, the amount of yellow coloration on fins and black coloration on beak, and all sperm quality traits were not affected by diet manipulations. Contrary to expectations, the area of red coloration on the beak and fins was lower in males maintained on the high compared with the restricted diet. Moreover, the pattern of resource-dependent expression of sexual traits was remarkably consistent between long-term diet restriction during development and short-term diet restriction after males reached sexual maturity, despite slight differences in experimental setup  – which was also contrary to our expectations. Overall, these findings highlight the complexities of understanding resource dependent expression of sexual traits.Our findings suggest that resource-dependent expression of sexual traits in male halfbeaks may influence their competitive ability. Most of the sexual traits that exhibit resource dependence in male halfbeaks have the potential to play a role in male–male competition. For example, male body condition and beak length are negatively influenced by resource limitations and these traits are likely to influence the outcome of competitive interactions. Males in better condition are often socially dominant and more likely to win male–male contests for access to mates or territories e.g., . Thus, rWe also detected patterns of resource-dependent expression of post-copulatory sexual traits that could influence the outcome of sperm competition in halfbeaks. Specifically, males experiencing resource limitations invested less in testes mass (controlling for body size). Testis size is correlated with sperm production rates and number of sperm cells produced , 1989 anResource restriction also impacted male courtship behaviors as male circling behaviors and mating attempts exhibited resource-dependent expression. These behaviors are a key part of male courtship and likely influence mate assessment and mating success . While aG. holbrooki; Gasterostaus aculeatus; An important finding from this study is the general consistency in the resource dependent response in sexual traits between the adult and juvenile diet manipulation experiments. Broadly speaking, this indicates that a relatively short diet restriction of one month during sexual maturity had a similar impact on sexual traits as a prolonged diet restriction of around 4 months until sexual maturation. In animals with indeterminate growth , sexually selected traits may be sensitive to resource availability throughout their lifetime, even after sexual maturity . Indeed,Resource-dependence in sexual traits appear less linked with mate choice in halfbeaks. For example, females were not influenced by the male diet treatment in their mate choice nor by differences between the males in their morphological traits. Since the males in the dichotomous trials were paired with no regard to their coloration, it may be that on average, there was low within-pair difference in morphology, making it difficult for the female to choose between the two males . On the more in the restricted-diet treatment in the case of red coloration on the beak and fins. The lack of reduction in coloration after diet restriction was unexpected, as the size and intensity of carotenoid-based coloration  is condition-dependent in many species  competition. An individual more capable of acquiring resources may display quality to potential mates through elaborate sexual behaviors and have higher fertilization success through larger sperm production capacities. The study also highlights the importance of incorporating many traits, as well as many aspects of the same trait (such as behavior), and a careful consideration of the timing of the resource manipulation in studies of resource dependence. Additionally, since sexual selection is a process that requires the influence of both sexes, future research should also investigate the resource dependence of traits in females, such as mate preferences and choosiness .arac060_suppl_Supplementary_Figure_S1Click here for additional data file.arac060_suppl_Supplementary_MaterialClick here for additional data file.arac060_suppl_Supplementary_Resource_1Click here for additional data file.arac060_suppl_Supplementary_Resource_2Click here for additional data file.arac060_suppl_Supplementary_Resource_3Click here for additional data file.arac060_suppl_Supplementary_Resource_4Click here for additional data file.


Enolase is an essential enzyme in the process of glycolysis and has been implicated in cancer progression. Though dysregulation of ENOs has been reported in multiple cancers, their prognostic value and specific role in bladder cancer (BLCA) remain unclear.Multiple databases were employed to examine the expression of ENOs in BLCA. The expression of ENO1 was also validated in BLCA cell lines and tissue samples by western blotting and immunohistochemistry. Kaplan–Meier analysis, ROC curve, univariate and multivariate Cox regression were performed to evaluate the predictive capability of the ENO1. Gene ontology (GO) and Gene Set Enrichment Analyses (GSEA) analysis were employed to perform the biological processes enrichment. Function experiments were performed to explore the biological role of ENO1 in BLCA. The correlation of ENO1 with immune cell infiltration was explored by CIBERSORT.By analyzing three ENO isoforms in multiple databases, we identified that ENO1 was the only significantly upregulated gene in BLCA. High expression level of ENO1 was further confirmed in BLCA tissue samples. Aberrant ENO1 overexpression was associated with clinicopathological characteristics and unfavorable prognosis. Functional studies demonstrated that ENO1 depletion inhibited cancer cell aggressiveness. Furthermore, the expression level of ENO1 was correlated with the infiltration levels of immune cells and immune-related functions.Taken together, our results indicated that ENO1 might serve as a promising prognostic biomarker for prognosticating prognosis associated with the tumor immune microenvironment, suggesting that ENO1 could be a potential immune-related target against BLCA.The online version contains supplementary material available at 10.1186/s12967-022-03509-1. Bladder cancer (BLCA) is a heterogeneous malignant tumor . There aKnown as the Warburg effect, tumor cells preferentially choose the glycolytic pathway to provide energy for cell growth, even under aerobic conditions, rather than the more productive oxidative phosphorylation pathway , 6. As tENOs are multifunctional molecules that not only participate in glycolysis pathway and regulate energy metabolism homeostasis, but also have a hand in the occurrence and development of various tumors . Recent Herein, we utilized bioinformatics analysis tools to explore the expression and multilevel clinical value of ENOs in BLCA, and identified ENO1 as a promising immune-related target, providing a novel strategy for the diagnosis and clinical treatment of BLCA.https://www.oncomine.org/resource/login.html) and TIMER database (https://cistrome.shinyapps.io/timer/) were utilized to quest the expression of ENOs between multiple cancer tissues and corresponding adjacent normal tissues. GEO database  was also used to analyze the expression difference of ENOs between BLCA tissues and normal samples. The association of the ENOs expression and clinicopathologic parameters was explored in UALCAN , TCGA-BLCA (https://www.tcga-data.nci.nih.gov/tcga) and GEO (GSE13507 and GSE32894) databases.Oncomine database  were applied, and scramble siRNAs (siNC) as the negative control. The siRNA sequences targeting ENO1 were listed in Additional file The human BLCA cell lines 5637 and UMUC-3 were cultured in RPMI-1640 medium (Invitrogen) at 37 °C with 5% COTotal RNA was isolated from the cultured cells using the TRIzol reagent (TaKaRa), RNA was then converted into cDNA by applying the Prime-Script RT-PCR kit (TaKaRa). The mRNA expression levels of genes were examined using SYBR Green in an ABI 7500 StepOne Plus Real-Time PCR instrument (Applied Biosystem). The specific primer sequences were listed in Additional file Western blotting was performed according to the standard methods as previously described . PrimaryThe stat packet of R software was employed to determine the co-expression genes associated with ENO1 expression in TCGA-BLCA. The clusterProfiler package of R software was utilized to perform GO function and KEGG pathway enrichment analysis of co-expressed genes.GSEA v4.1.0 software was applied to perform GSEA to investigate meaningful biological processes associated with ENO1 expression. Pathways with nominal p-value < 0.05 and FDR < 0.25 were considered significantly enriched.Survival analysis was performed using the survival package and survminer package of R software to draw the Kaplan–Meier curves on BLCA samples. Survival package was also applied to perform univariate and multivariate Cox analysis, and survivalROC package was utilized to draw ROC curves.2,000 cells/well were seeded into a 96-well plate. If cells adhered to the bottom, 10 μL MTT was added to each well for 4 h at 37 °C and it was identified as 0 h. The formazan crystals were dissolved in dimethyl sulfoxide (DMSO) at 37 °C for 15 min and the absorbance at 490 nm was examined. After 24, 48, and 72 h, the similar procedure was performed.5 cells were seeded into the top of an 8 μm pore-size Transwell chamber pre-coated with diluted Matrigel (BD Biosciences), then 500 μL medium containing 10% FBS was added to the bottom chamber. After the incubation for 24 h, cells were fixed in formaldehyde, stained with crystal violet, and counted by applying a microscope.A total of 1 × 10CIBERSORT package of R software was used to detect the proportion of 22 immune cells in BLCA samples with low and high ENO1 expression, and Pearson’s correlation was assessed between the proportions and ENO1 expression.To investigate the prognostic worth of ENO isoforms in BLCA patients, we firstly utilized Oncomine database to analyze the expression of ENOs. As shown in Fig. Next, UALCAN database was applied to examine the association between ENOs expression and clinicopathologic characteristics in BLCA patients. As shown in Fig. To further quest the clinical significance of ENOs in BLCA, the correlation of ENOs expression and prognosis was examined by means of Kaplan–Meier survival analysis. The results manifested that ENO1 expression was negatively associated with overall survival in all 3 databases , which mainly consists of the stromal cells, blood vessels, extracellular matrix and lymphatic networks, plays a significant role in cancer initiation and progression, angiogenesis, and even immune escape –31. The To examine the interaction of ENO1 in TME, CIBERSORT algorithm was utilized to analyze the difference in the proportions of immune cells in the two groups with high and low ENO1 expression. The results revealed that 8 of 22 immune cell types showed significant differences with ENO1 expression Fig. A. FurtheBladder cancer (BLCA) is one of the most common malignancies in the genitourinary system . Its higHence, to demonstrate the potential worth of ENOs in BLCA, the expression and clinical prognostic value of ENOs were analyzed. First of all, we were pleasantly surprised to find that the expression level of ENO1, but not other ENO isoforms, was significantly up-regulated at the mRNA level in BLCA in Oncomine, TIMER, UALCAN, TCGA-BLCA and GSE13507 databases. Western blotting and immunohistochemical further demonstrated aberrant overexpression of ENO1 at the protein level. Subsequently, the clinical prognostic value of ENO1 was explored. High expression of ENO1 was prominently correlated with high pathological grade and advanced clinical stage. Moreover, overexpression of ENO1 predicted worse prognosis in patients with BLCA. ROC curves also showed that ENO1 had significant diagnostic value for BLCA. Meanwhile, nomogram model illustrated that ENO1 could serve as an independent prognostic factor, which could be utilized to estimate the prognosis of patients. Of particular note, few studies have also elevated the circulating level of ENO1 in cancer patients. For example, ENO1 was overexpressed in the plasma of patients with pancreatic cancer, and the increased plasma ENO1 level was correlated with prognosis and disease progression . In addiThe current research on the function of ENO1 in tumor is primarily focused on its effects in glycolysis, while comprehensive analysis of ENO1 in BLCA is less studied. We first identified genes that were significantly correlated with ENO1by constructing a co-expression network, among which TPI1, RAN and GAPDH showed the strongest correlation with ENO1 in BLCA. Previous studies have demonstrated that TPI1 and RAN exerted crucial effects on tumor initiation and progression –40. For Increasing evidence has demonstrated that infiltrating immune cells in the tumor microenvironment plays a crucial role in tumorigenesis and progression, thereby affecting the prognosis of tumor patients –45. In tChanges in tumor metabolism provide new therapeutic targets for tumor therapy. As mentioned earlier, genes related to the glycolytic pathway have been the focus of tumor target research. As the core catalytic enzyme of glycolysis, increasing studies have begun to look for molecules to effectively inhibit ENO1 activity. Previous studies have found that PhAH was a pan-enolase inhibitor, which could effectively inhibit the activity of ENO1, thereby suppressing the growth of pancreatic, breast, and lung cancers , 46. AnoAlthough the present study initially revealed the association of ENO1 with BLCA, some limitations still exist. Firstly, we confirmed abnormally high ENO1 expression in BLCA at both mRNA and protein levels. However, further studies should be conducted to investigate the specific role and potential molecular mechanisms of ENO1 in tumorigenesis, progression, and immune infiltration. As a metabolite of ENO1 during glycolysis, the biological role of phosphoenolpyruvate in BLCA has also not been fully evaluated, enlightening us that further exploration should be attached to the biological significance of the metabolites correlated with ENO1. In addition, most of the analyses were performed based on TCGA and GEO cohorts, which lack further experimental validation. More in-depth exploration to explain these findings should be systematically interpreted in vitro and in vivo to make the results more convincing.Overall, our results revealed that ENO1 was abnormally overexpressed in BLCA, upregulation of ENO1 predicted unfavorable clinical outcomes and was significantly associated with tumor malignancy. These outcomes implied that ENO1 might serve as a promising prognostic biomarker for BLCA. Additionally, ENO1 expression was remarkably related to immune cell infiltration in the TME of BLCA. Taken together, these findings help to deepen our understanding of not only the effects of ENO1 but also its translational application in diagnosis and therapy of BLCA.Additional file 1: Table S1. List for sequences of primer sets and siRNAs.Additional file 2: Figure S1. Prognostic analysis of ENO1 in the BLCA patients. A, B Univariate (A) and Multivariate (B) Cox regression analyses of ENO1 along with clinicopathological characteristics for overall survival in GEO13507. C Time-dependent ROC analysis of ENO1 in estimating the prognostic performance of the BLCA patients in GEO13507. D Multi-variable time-dependent ROC analysis of ENO1 in predicting the overall survival of the BLCA patients in GEO13507.Additional file 3: Figure S2. The originally western blotting images were presented.


In Western blot analysis, conjunctival AQP5, MUC5AC, and CFTR expression increased significantly in response to DS, compared to control . Immunohistochemistry for CD31 and LYVE-1 were performed. CD31-positive cells and lymphatic space surrounded by LYVE-1-positive cells increased significantly in conjunctival tissue post-DS, compared to control . Surface damage was worse in the corneal than in the conjunctival epithelium after DS, by scanning electron microscopy. This study showed that the cornea and conjunctival epithelium show differences in vulnerability to DS. Increased blood vessels and dilated lymphatics, accompanied by increased conjunctival epithelial AQP5, MUC5AC, and CFTR expression, underlie the protective mechanism of the conjunctiva to desiccating stress.We evaluate the difference in vulnerability to desiccating stress (DS) between the corneal and conjunctival epithelia to understand different ocular surface staining patterns in dry eye patients. We generated a rabbit model of short-term exposure keratopathy. To induce DS in the ocular surface, rabbit right eyelids were opened for 30 min, with blinking once/minute. Corneal staining scores increased from 3-min post-DS exposure, while conjunctival staining increased from 20-min post-DS. At 20 min, the tear MUC5AC level doubled as compared to pre-DS ( The presence of ocular surface staining represents corneal epithelial barrier disruption and/or conjunctival epithelial damage2. Moreover, the nature and pattern of punctate ocular surface staining in DED provides useful clinical information3. Various ocular surface scales have been developed to assess ocular surface states. These employ different methods to score ocular surface staining: some include the conjunctiva, while others include only the cornea7. Although epitheliopathy is commonly observed in DED and contributes to tear film instability8, some DED patients demonstrate no ocular surface staining, and only corneal staining without conjunctival staining, or vice versa, which implies that the staining patterns of the cornea and conjunctiva derive from different mechanisms10. A study by Yang et al.11 reported that conjunctival staining scores may be useful to measure ocular surface inflammation in DED, while corneal staining scores did not show any relationship with mRNA levels of various inflammatory cytokines. Ocular surface staining with fluorescein is extensively used to highlight corneal and conjunctival damage in the diagnosis of dry eye disease (DED)12. Several animal models have been developed to obtain a deeper understanding of the DED inflammation process13. Other models of ocular surface disease have been developed that do not necessarily involve ocular surface inflammation or desiccation but instead resort to more subtle changes, like hyperosmolar stress15. These are categorized into the aqueous-deficient and the evaporative types induced by mechanical or surgical approaches, iatrogenic immune responses, topical eye drops, or blockage of neural pathways18. A study by Lai et al. in 201519 first reported ocular surface changes in a rabbit model of exposure keratopathy. Whereas the previous studies focused on confirming ocular surface inflammation in DED models, in the present study, to gain understanding of the differences in staining patterns of cornea and conjunctiva in DED, we generated a rabbit model of short-term exposure keratopathy and focused on the ocular surface compensatory mechanism against desiccating stress. We aimed to investigate the difference in vulnerability to desiccating stress between the cornea and conjunctival epithelium. This allowed us to investigate the protective effect of the ocular surface, excluding for the protective effect of the eyelids, against desiccating stress.Over the past two decades, growing evidence has shown that inflammation affects the ocular surface in DEDp = 0.0023, Friedman test, Fig. p = 0.0003, Friedman test, Fig. p = 0.0002, Friedman test, Fig. During the 30-min exposure, rabbit eyes were evaluated at each time point: 3 min, 10 min, 20 min, and 30 min. Conjunctival hyperemia continued to increase after 3 min of exposure and reached the highest score after 30 min  and lymphatic vessel endothelial hyaluronan (HA) receptor 1 . The number of CD31-positive cells was significantly increased after ocular surface exposure in conjunctival tissue, as compared with control tissue  upregulate MUC5AC mRNA expression27. This should be distinguished from the chronic disease condition, in which the goblet cell number and MUC5AC concentration are decreased, indicating that the compensatory mechanism is compromised26.Mucin plays important roles in the ocular surface and can be classified broadly according to its structural characteristics as secreted mucins and membrane-associated mucins29. AQP5 is typically expressed on the apical membrane of the acinar and ductal epithelial cells in human lacrimal glands30. AQP3 and AQP5 have also been identified in conjunctival epithelium31. In 2015, Bhattacharya et al. reported the pivotal role of the conjunctiva in the maintenance of ocular surface homeostasis in rabbits with bilateral resection of the main lacrimal gland and reported that AQP4 and AQP5 are possibly involved in restoring the ocular surface fluids33. The expression of AQP5 and MUC5AC has been either positively or negatively correlated in the respiratory track epithelium in different reports36. Similarly, several studies have reported that close regulatory mechanisms exists between AQP5 and MUC5AC at the ocular surface. In the present study, expression of both AQP5 and MUC5AC were significantly increased in the exposure model as compared to in the control eyes, which supported the findings of previous studies.Aquaporins (AQPs) are a group of transmembrane water-channel proteins that mediate the passage of water molecules38. Several studies have reported DED in patients with cystic fibrosis, suggesting the potential influence of CFTR in tear secretion43. Recently, Berczeli et al. reported decreased tear secretion and impaired ocular surface integrity in CFTR-knockout mice38. In our study, the expression of CFTR in the conjunctival epithelium was significantly increased in eyes exposed to short-term desiccating stress as compared to control eyes, which implies transient increase in fluid efflux as a compensatory mechanism of the conjunctiva to desiccating stress.Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel expressed in various secretory epithelia, including airways, gastrointestinal organs, salivary glands, sweat glands, and the ocular surface. When defective, it may cause cystic fibrosis, the most common genetic disease among Caucasians45. Recently, there has been growing recognition of the importance of corneal lymphatic vessels in mediating the activation and movement of immune cells at the corneal surface in DED47. This recognition has grown significantly with the development of reliable lymphatic markers. Lymphatic vessel endothelial hyaluronan (HA) receptor 1 (LYVE-1) is a member of the Link superfamily of HA-binding proteins that is highly expressed on lymphatic vessels and serves as a lymphatic-specific marker50. In the present study, the lymphatic space surrounded by LYVE-1-positive cells was significantly increased in conjunctival tissue of eyes after ocular surface exposure as compared to in the control tissues. The number of CD31-positive cells was also increased after short term desiccating stress. CD31 is an endothelial cell surface marker, indicating blood vessels, whereas LYVE-1 functions in cell adhesion/transmigration in lymphangiogenesis46. These results indicate that dilatation of lymphatic and blood vessels could be an early response after desiccating stress and suggest that lymphangiogenesis along with hemangiogenesis could form part of a compensatory mechanism to maintain homeostasis of the ocular surface.Lymphatics play an important role in generating immunoinflammatory responses in peripheral tissues by directing antigen-presenting cells from the periphery to the draining lymph nodesThere are several limitations in this study. First, this study used a small number of animals to draw conclusions. However, the results were consistent in showing the difference between the cornea and conjunctiva and revealing the protective effect of the conjunctiva. Second, the use of normal saline in the process of tear sample collection could have affected the ocular surface of rabbit models by a temporary wetting effect. To minimize this effect and obtain consistent results, we standardized the experimental conditions by using a set sequence of examinations. Third, we only evaluated up to a 30-min exposure, which does not reflect the effects of long-term exposure of the ocular surface. However, since our main purpose was to confirm the protective effect of the conjunctiva or cornea against desiccating stress, we consider that this was sufficient time to confirm a short-term protective effect. Last, we could not evaluate the recovery of the ocular surface over time after short-term exposure.In conclusion, in this study, we demonstrated a difference in vulnerability to desiccating stress between the corneal and conjunctival epithelium and confirmed that this vulnerability difference causes the difference in corneal and conjunctival staining in dry eye patients. The increase in blood vessels and the dilatation of lymphatic vessels in the conjunctival tissue, accompanied by increased expression of AQP5, MUC5AC, and CFTR in the conjunctival epithelium, seem to underlie the protective mechanism of the conjunctiva to desiccating stress. Further studies with models such as Sjögren’s syndrome and graft versus host disease, in which the compensatory mechanism of the conjunctiva is damaged, will be helpful to better understand the compensatory mechanism of conjunctiva.New Zealand white rabbits  were used in this study. The study was carried out in compliance with the ARRIVE guidelines. All procedures adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research . Approval for this study was obtained from the Korea University Guro Hospital Institutional Review Board, Seoul, South Korea (KOREA-2021–0122).50. The right eyes of the rabbits were used for all experiments, and the left eyes were left untreated and were used as controls. The interpalpebral fissures were kept wide-open for 30 min to ensure the exposure of the central cornea, limbus, and perilimbal conjunctiva, with blinking performed manually once every minute. Eyes were evaluated at each time point: 3 min, 10 min, 20 min, and 30 min.All in vivo experimental procedures  were performed under general anesthesia induced by the intramuscular injection of ketamine hydrochloride (35 mg/kg) and xylazine hydrochloride (5 mg/kg)The cornea and conjunctiva of rabbits were examined under a portable slit-lamp microscope with a cobalt blue filter, under general anesthesia, after instillation of 1 drop 1% fluorescein solution. A yellow filter was used to evaluate the staining of the conjunctiva in detail. For corneal fluorescein staining (CFS) scores, fluorescein sodium-impregnated paper strips  were applied to the upper bulbar surface after retracting the upper eyelid and after wetting the end of the strip with 5 μL saline.4. The conjunctival hyperemia was graded using the Efron scale for conjunctival redness, which consists of five grades (0–4)51.The ocular staining score was assessed by a single experienced ophthalmologist according to the standard National Eye Institute (NEI) grading system. Briefly, the cornea was divided into five areas ; punctate fluorescein staining in each area was graded on a scale of 0 to 3, and the total score (0–15) is the sum of scores for all five areas. The conjunctiva was divided into six areas and the total score (0–18) is the sum of scores for all six areas52. Tear samples were used to measure MUC5AC levels, to investigate conjunctival goblet cell mucin secretion into the tears. The level of MUC5AC in the tear sample was assessed using an enzyme-linked immunosorbent kit for rabbit MUC5AC . All measurements were conducted according to the manufacturer’s protocol, using a microplate spectrophotometer .Tear samples were collected from the rabbit eyes baseline and after 3, 10, 20, and 30 min of exposure of the conjunctiva and cornea. Sixty microliters of normal saline was gently applied to the inferior fornix using a micropipette, twice. Thereafter, a total volume of 120 μl of a tear sample was gently collected53.After the last tear sample was collected after 30 min of eye exposure, corneal and conjunctival impression cytology was performed. After instilling an 0.5% proparacaine hydrochloride eye drop . An 8-mm-diameter nitrocellulose membrane  was applied to the cornea and conjunctiva. The membrane was then gently peeled off with smooth forceps. The membrane was immediately immersed into a well that was filled with fixing solution. Hematoxylin and eosin (H&E) and periodic acid‒Schiff (PAS) staining were used for histological staining of the membrane in the specimenTissue cell extracts from corneal impression cytology were subjected to western blot analysis to measure protein levels of AQP5. Those from conjunctival impression cytology were subjected to western blot analysis to measure protein levels of AQP5, MUC5AC, and CFTR. Primary antibodies against AQP5 , MUC5AC , CFTR , and β-actin  were used. The impression cytology from left eyes, which left untreated were used as controls. The original images as they were saved during the experiment are presented in Figure S1–S4 as supplementary files. We only stored the protein band images of interest and images of full-length blots were not presented.54. We defined the percentage of cell loss as the ratio of the cell loss, which shows epithelial cell sloughing or morphologic change to the total epithelial cells in each four representative digital images from SEM to quantify the epithelial cell loss. First, total area of each representative images was calculated using ‘set scale’ based on the known distance from SEM using ImageJ . Second, area of each corneal and conjunctival epithelial cell was calculated from ‘polygon selection tool’. Third, count of total epithelial cells in each representative image was calculated as the total area of each representative images divided by the area of each epithelial cell. Fourth, the number of cell loss, which shows epithelial cell sloughing or morphologic change was calculated using ‘multi-point tool’  and immunohistochemistry, rabbits were euthanized using a CO2 chamber under general anesthesia. The corneal and conjunctival tissues were gently excised after sacrifice and were prefixed in 2% glutaraldehyde in 0.1 M phosphate buffer. Samples were then post-fixed for 2 h in 1% osmic acid dissolved in phosphate-buffered saline for SEM. Then, the corneal and conjunctival tissues were treated in a graded series of ethanol and t-butyl alcohol, dried in a freeze dryer , and coated with platinum using an ion coater . The appearance of the corneal and conjunctival epithelial surface was observed via field emission-SEM The anterior segment of each eyeball was surgically removed and fixed in 10% neutral buffered formalin and was then embedded in paraffin. Paraffin-embedded tissues were cut into 4-μm sections with a microtome , and tissue sections were placed on microscope slides. After deparaffinization of the tissue sections with xylene, tissue sections were immersed in a graded series of ethanol and phosphate-buffered saine. Serial sections were used for immunohistochemistry of CD31  and LYVE-1 . Primary antibodies were commercially obtained for CD31 , and LYVE-1 . A rabbit-specific HRP/DAB (ABC) Detection IHC Kit  was used for secondary antibody-based detection according to the manufacturer’s instructions. The tissue sections were observed under light microscopy at 400 × magnification, and digital images were taken with an Olympus BX51 microscope and a DP72 camera . The cells positive for CD31 were determined by analyzing four visual fields on conjunctival biopsy and results are reported as cells per square millimeter. The lymphatic space surrounded by LYVE-1-positive cells (%) were defined as percentage of the summed area surrounded by LYVE-1 positive cells per total area observed. The mean CD31-positive cell count and the lymphatic space surrounded by LYVE-1-positive cells (%) were compared between the exposure model eye and the control eye.P < 0.05 was considered statistically significant.Statistical analyses were performed using the Mann‒Whitney U test and Wilcoxon signed-rank test in SPSS version 20.0 . Values are expressed as the median and interquartile range. Supplementary Information.


Wheat germ has been recognized as an economical source of high-quality plant proteins and bioactive compounds for food fortification. Thus, it can be used for valorization of food products as a feasible strategy to enhance the nutritional quality and reduce wheat milling waste. In this research roasted wheat germ (RG) was added in formulation of egg-free milk pudding to enhance its nutritional value and the effects of RG particle size  and quantity  on the quality, nutritional and sensory properties of the resulting pudding were investigated. Reducing the particle size of RG significantly altered its chemical composition but had no significant effect on its antioxidant activity. Increasing the level of RG in the pudding, reduced pH and syneresis while increased dry matter content, hardness, cohesiveness and gumminess of the product. The quantity of RG had more effects on physicochemical properties of the puddings than changing the particle size. Based on the sensory evaluation results, the most acceptable sample was obtained by addition of 7.5% RG with a particle size of 125 μm. The demand for functional foods with documented health benefits is growing rapidly, since they can offer a unique opportunity to transform our daily foods into affordable health promoting delivery vehicles for the growing population ,2. HowevGrains are economical and rich sources of bioactive compounds, proteins and nutrients required for a healthy life, to combat the rapid increase in chronic diseases as well as mineral deficiency, known as hidden hunger around the world. However, grain processing such as milling results in grain loss specially the loss of nutrient dense parts including germ and bran. Therefore, employing practical strategies to reduce grain wastage and valorisation of the by-products are of great importance in fighting food waste and improving the nutritional value of foods at an affordable cost. Wheat germ is an economical and nutrient dense by-product of wheat milling factories. In 2012, about 120,000 tons of wheat germ were produced from wheat milling with an anticipated increase in the recent years due to higher wheat production and consumption . Wheat gDespite the benefits of wheat germ, its applications as a food ingredient are limited and it is mainly wasted as animal feed. It is mostly attributed to its high unsaturated lipids and enzyme activity resulting in rapid rancidity issues including off-flavour, reducing nutritional quality and shelf-life of the wheat germ ,7,13. ThTo overcome wheat germ limitations in food industry, dry heating (roasting) can be applied as an available and practical thermal processing method to inactivate enzymes and reduce moisture content as well as enhancing colour and flavour in wheat germ . RoastinThe enrichment of bakery products such as bread, cake and cookies with wheat germ has been reported ,15 whileMilk pudding is a popular product around the world amongst children, elderlies, athletes and health-conscious consumers . Thus, aThe major objective of this research was to produce a novel value-added milk pudding using wheat germ to improve the nutritional value of the pudding and also to decrease wheat wastage during milling. Viable strategies  were applied to combat some of the shortcomings of value addition process including loss of sensory attributes and quality failure. Raw wheat germ was supplied by Sepidan Wheat Milling Factory . Pudding ingredients including gelatine powder was obtained by Behin Azma , white fine sugar and fresh pasteurized skim milk with 1% fat content were purchased from a local supermarket. Analytical grade chemicals used in this research were obtained from Merck Company .To stabilize wheat germ, a thin layer of raw wheat germ (1 cm thickness) was spread over a perforated aluminium tray and roasted at 180 °C for 15 min in an electrical oven. It was then cooled down to room temperature for 1 h. This method can result in high wheat germ stability ,18. The RG was ground once in an Gosonic grinder  and sieved through stainless-steel sieves  to obtain different particle sizes. Preliminary tests showed that RG can be readily ground to the average particle sizes of 125, 210 and 354 μm while achieving smaller particle sizes was not practical. Ground RG samples were packed in sealed plastic bags and kept at 4 °C. Approved methods of AACC (2000) were used to determine chemical composition of RG  . Extraction of polyphenols was conducted based on the method using methanol (99.5%) . Alumini2CO3  was added to the sample and incubated for 45 min at ambient temperature. Then the absorbance was measured at 765 nm after using a UV/visible spectrophotometer . A calibration curve of gallic acid  was obtained, and the total phenolic content  was measured. Antioxidant activity of the methanol extract was measured based on scavenging activities of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.Folin–Ciocalteu’s reagent method was used to determine the total phenolic content . Samplesw/w) of RG. Preliminary tests showed poor sensory properties of the puddings made with more than 10% RG as they became dark and very thick and hence was not tested. To prepare puddings, RG was first hydrated in 1/3 of the milk and stored at 4 °C for 2 h. The remaining milk was heated up to about 50 °C in a sealed glass container and then gelatin powder and sugar were added slowly and blended for 5 min . The hydrated wheat germ in milk was added to the mixture, sealed and batch pasteurized at 72 °C for 30 min. The ingredients of the pudding were 10% white sugar, 2% gelatin powder, 88% skim milk (cow milk) and different particle sizes  and concentrations  and capped to prevent dryness. The samples were stored at 4 °C for 24 h (fresh samples) and 15 days (stored samples) before texture analysis tests. For other tests including sensory tests, 68 g of the hot mixtures were transferred into plastic cups , hermetically sealed and refrigerated for 24 h at 4 °C. The dry matter was obtained by deducting the moisture content (obtained by oven drying till a constant weight obtained) from 100. w/w) and the pH was recorded using a pH meter  after calibration. To measure pH, the pudding was diluted in distilled water 1:2  using a texture analyser  using a 30 kg load cell. Two successive compressions were applied on the samples using a cylindrical probe (40 mm diameter) to compress the samples to 25% of their heights with an interval time of 10 s. The crosshead speed was maintained at 0.25 mm/s. The trigger force (6 g), pretest speed (5 mm/s), test speed (0.25 mm/s) and posttest speed (5 mm/s) were constant for all samples.The texture profile parameters including hardness, springiness (elasticity), cohesiveness and gumminess were derived from the force–distance curve .Digital colourimetry method was used to puddings colour parameters . DigitalTo determine syneresis, the samples were removed from the plastic cups  using a 5-point hedonic. The sensory attributes were described by a liking score (where 1 = strongly disliked to 5 = strongly liked). The samples were removed from refrigerator, emptied on transparent plastic plates with random codes and presented to the panellists is standard isolated booths. The colour of the samples was evaluated under day light while other sensory attributes under red light . A small piece of each freeze-dried pudding sample was sticked onto an aluminium sample holder and coated with a thin layer of gold metal using an ion sputter coater  and examined using a scanning electron microscope  with a working distance of 7.5–9.5 mm and at 20.0 kV.p < 0.05) using Statistical Analysis System (SAS) software, version 9.3.1. A completely randomized design was used for analysis of the triplicates data. Average and standard deviation were calculated using Excel 7. Significant differences between the average values obtained for different characteristics of the puddings due to the various concentrations and particle sizes of RG and storage time were evaluated using analysis of variance techniques (ANOVA) and Multiple Ranges Duncan’s test was used to differentiate between the means (50) varied between 54.30 and 54.35. The results revealed that changing the particle size had insignificant consequence on total flavonoid content and IC50 of RG; however, reducing the particle size slightly reduced the total phenolic content from 5.84 to 5.06 mg gallic acid/g. This may be due to the heat generated during milling process (used for reducing the particle size of the germ) casing degradation of the phenolic compounds, however, further studies are required to confirm it. The phenolic and flavonoid contents of the RG were 5.06–5.84 mg/g and 5.32–5.54 mg/g, respectively and the antioxidant activity (ICCompared to the unroasted wheat germ (raw germ) as tested in our previous study , the roaAs shown in As p < 0.05) on the textural parameters of the samples. The micrographs of the samples  with addition of RG level, while it remained unaffected by changing the wheat germ particle size. The decrease in syneresis of the RG samples can be related to the high amount of carbohydrates, protein and fibre content of the germ which can form hydrogen bonds with water molecules due to their large number of hydroxyl groups and hinder water separation from the samples. Similar results were reported for chilled dairy dessert containing raw wheat germ [Generally, lower syneresis is desirable in puddings as it affects shelf-life, sensory aspects and customer acceptance of the products. Based on the results  the wateeat germ . As This research emphasises the feasibility of wheat germ utilization as an economical plant-based functional ingredient in production of milk pudding to enhance its nutritional value and also as a successful upcycling strategy to reduce food waste in wheat supply chain. The main effects of changing the particle size of wheat germ were observed on the chemical composition of wheat germ and dry matter and colour of the puddings, while other pudding properties including pH, texture, sensory properties and syneresis were not significantly affected. Large wheat germ particles  provide higher protein, fat and fibre content but almost similar amounts of flavonoids and antioxidant activity compared to the small size particles (210 µm). This information may be useful in product formulation to adjust the chemical composition of the end product if required. The quantity of the added RG was an important factor influencing the milk pudding quality. Wheat germ can be added up to 10% to enhance dry matter, textural hardness, reduce syneresis and improve sensory properties of the puddings. However, pudding colour became caramel brown by addition of wheat germ. If this becomes an issue, addition of natural and approved food colorants such as caramel, cocoa powder or chocolate may be useful. Generally, pudding quality was more significantly affected by wheat germ concentration than the size of particles. Addition of 7.5% RG with a particle size of 210 µm resulted in the most acceptable sample. This can add 2.67 g protein, 0.77 g fat, 0.34 g ash, 0.55 g fibre, 40.65 mg/g total phenolic content and 41.92 mg/g total flavonoid content to 100 g of milk pudding and hence improve the nutrition profile and antioxidant content as compared to ordinary milk pudding.


Fusarium is a phytopathogenic fungus involved in human pathology and is present in space stations. It is essential to understand the effects of microgravity on the physiology of this fungus to determine the potential risks to the health of crew members and to propose the necessary countermeasures. This study aimed to determine changes in the physiological parameters of the Fusarium solani species complex under simulated microgravity generated using a random positioning machine (RPM) and phenotypic approaches. We observed increased growth, spore production, and germination while biofilm production was reduced under RPM exposure. These in vitro data show the importance of further studying this fungus as it has been repeatedly demonstrated that microgravity weakens the immune system of astronauts. Fusarium has been described in surface samples and condensation liquids of the Mir (Russian space station) and ISS stations [Fusarium strains have been isolated on the surface of the ISS dining table but also in lesions observed on a plant cultivated at this station [Fusarium when it grows under simulated microgravity. On Earth, the Fusarium genus is a transkingdom pathogen. It is a phytopathogenic fungus but also an emerging fungus involved in the origin of invasive fungal infections in immunocompromised human subjects, characterized by fungemia or skin lesions [Fusarium solani species complex (FSSC) represents 60% of human fusariosis in humans [The space environment modifies numerous human physiological functions. A recent study of medical data collected from 46 astronauts who spent 6 months on the International Space Station (ISS) showed that 46% encountered immunological problems such as hypersensitivities and infections . The ISSstations ,4. Recen station ,6. The p station ,8,9. Thi station . It is t lesions . The Fusn humans .Candida albicans [Some studies have analyzed the adaptation of fungi to microgravity , but thealbicans . Phenotyalbicans .Fusarium solani species complex. Indeed, it is essential to understand these effects to determine the potential risks to the health of crew members and to propose the necessary countermeasures. To our knowledge, no data are currently available for this pathogen. In a first descriptive approach, essential given the current lack of knowledge, we evaluated the physiological parameters of growth, germination, and ability to generate biofilms under RPM exposure.Due to its presence in space stations and its major pathogenicity in humans, we studied the effect of simulated microgravity on the physiological aspects of the FSSC isolates  were used for this study  , recommended to describe croscopy  and Rosw4 conidia/mL and incubated at 30 °C in the dark. The growth was evaluated by optical density measurements at 600 nm for 24 h in microplates. Each well (RPM and static 1 g) was incubated with 5 × 106 conidia/mL. After 24 and 48 h of incubation at 30 °C, for each condition (RPM and static 1 g), the number of colonyforming units (CFUs) was counted on Sabouraud agar . To evaluate the spore production between the two gravity conditions, 5 mL tubes of liquid Sabouraud medium  were inoculated with 10The experiment was performed twice for all 8 strains.g and conidia were counted on Kova slides , and diluted in liquid Sabouraud medium to 106 conidia/mL [For the germination assay, conidia were collected by adding 5 mL of sterile water containing 0.1% Tween 80 to the Petri dish after peeling conidia off three times with a rake. The recovered suspension was centrifuged for 5 min at 2000× nidia/mL . Each 5 6 conidia/mL produced in Sabouraud medium was injected into the device followed by 25 mL of Sabouraud culture medium. The chamber was then incubated for 90 min at 37 °C flat and without shaking for cell adhesion. Then, the cultures were incubated for 72 h at 37 °C in the dark under either RPM exposure or 1 g control conditions. The media was removed, and pieces of the chambers (1 cm2) were cut for quantitative and qualitative analysis of the biofilm. These analyses were performed for the CBS 124630 strains in duplicate and the experiment was performed five times.Petaka culture chambers were used to carry out fungal cultures in liquid medium without air bubbles . It is a rectangular chamber (85.5 × 127.5 × 5.0 mm) designed for cell culture that contains an automatic gas diffusion device. For biofilm formation, 4 mL of standardized cell suspension at 10g. The supernatant was collected and its absorbance was measured at 490 nm.A semiquantitative approach to biofilm synthesis was carried out using the XTT -2H-tétrazolium-5-carboxanilide) method described by Chandra . Petaka A qualitative approach to biofilm synthesis was carried out by confocal microscopy according to Chandra’s protocol . It invohttps://www.xlstat.com, 1 March 2021). Differences between groups were determined using Student’s t tests and considered statistically significant at p values ≤ 0.05.Data analysis was performed using XLSTAT 1194 .A growth difference was observed at 24 h of culture between the 1 g control (OD of 0.563) and RPM exposure (OD of 0.670) conditions (p < 0.001). At 48 h of incubation, the number of colonies was 111 and 46, respectively, under the conditions described above (p < 0.001).Under simulated microgravity, spore production was twice as high as in the 1 g control condition . Indeed,p < 0.01); at 48 h, the values were 84 and 91%, respectively (p < 0.01). Under RPM exposure, multiple germination tubes on several areas of the spore were observed, while they were absent in the control condition . Confocal microscopy observation showed that the biofilm formed in 1 g controls was denser than the ones formed under RPM exposure for each experiment . The thiFusarium, a filamentous fungus that has already shown pathogenicity in plants in space conditions [Fusarium has been associated with material degradation [This study aimed to understand the impact of microgravity, simulated by an RPM, on the physiological parameters of nditions ,6. Furthradation .Experimentation in real microgravity is expensive and scarcely available; thus, a variety of platforms with ground-based facilities provide experimental conditions on Earth comparable to real microgravity . In thisPleurotus ostreatus, macroscopic modifications of fruiting body development are obvious under simulated microgravity. Growth against the direction of gravity is observed when the fungus is placed under static conditions (fixed to the ground), whereas the mushroom fruits radially under simulated microgravity [Aspergillus niger, Cladosporium herbarum, Ulocladium chartarum, Basipetospora halophila), whose growth took place onboard the ISS [For the mushroom ogravity . In our  the ISS . For yea the ISS , whereas the ISS .Candida albicans, Saccharomyces cerevisiae and Aspergillus fumigatus under spaceflight or simulated microgravity conditions [In our study, we showed an increase in fungal growth and spore production under RPM exposure. Comparable data were described for nditions ,20,23,24Aspergillus niger and Penicillium, whereas no differences were observed for Aspergillus fumigatus [Candida albicans, most studies showed an increase in yeast filamentation [Candida albicans, Saccharomyces cerevisiae) with a higher frequency of multiple buds and sometimes the formation of yeast clusters [We also observed an increase in spore germination under RPM exposure with spores presenting multiple germination tubes. The same observation was made for umigatus ,37. For entation ,16,17,19clusters ,15,22,23Fusarium biofilm formation under RPM exposure while Candida albicans grown under simulated microgravity presented a more complex biofilm [By two complementary approaches, we demonstrated a decrease in  biofilm . However biofilm .Fusarium solani presents stronger growth and greater germination, which raises the hypothesis of a greater fungal load in pathology. On the other hand, it appears that this fungus has less need to resort to its biofilm form in this environment, suggesting better accessibility for immune cells or antifungal molecules. To test these hypotheses, studies of virulence and susceptibility in an in vivo model are needed. This type of study is essential in the field of space research to establish the necessary countermeasures to preserve the health of the crew as well as the integrity of the installations. In addition, in the medical field, these models allow us to understand the potential modifications of microorganisms in low shear areas of the body such as microvilli of epithelial cells in the gastrointestinal, urogenital, and respiratory tracts [Thus, our study showed that under simulated microgravity conditions, the filamentous fungus y tracts ,38.Fusarium solani.To conclude, we have shown that simulated microgravity prompts physiological changes in the filamentous fungus