During unpleasant imagery, muscle lengthening started at 59.00 ± 22.50% of maximal AF, in contrast to baseline and pleasant imagery, during which the isometric position could be maintained mostly during the entire force increase up to ~97.90 ± 5.00% of maximal AF. Healthy participants showed an immediately impaired holding function triggered by unpleasant imagery, presumably related to negative emotions. Hence, AF seems to be suitable to test instantaneously the effect of emotions on motor function. Since musculoskeletal complaints can result from muscular instability, the findings provide insights into the understanding of the causal chain of linked musculoskeletal pain and mental stress. A case example (current stress vs. positive imagery) suggests that the approach presented in this study might have future implications for psychomotor diagnostics and therapeutics.The link between emotions and motor function has been known for decades but is still not clarified. The Adaptive Force (AF) describes the neuromuscular capability to adapt to increasing forces and was suggested to be especially vulnerable to interfering inputs. This study investigated the influence of pleasant and unpleasant food imagery on the manually assessed AF of elbow and hip flexors objectified by a handheld device in 12 healthy women. The maximal isometric AF was significantly reduced during unpleasant vs. pleasant imagery and baseline ( The interaction between emotions and motor control has been discussed for decades, especially in psychology and behavioral science  and imax) is exceeded, but the force increases further during the eccentric phase. The maximal AF of the trial refers then to the maximal eccentric AF. Hence, the maximal AF (AFmax) can be achieved either during isometric or eccentric muscle action. In case of a stable adaptation, AFisomax is similar or considerably high related to AFmax or to MVIC, respectively [max), whereby AFmax reaches a similarly high level as for stable adaptations but then during eccentric motion [max was found to be significantly reduced in healthy participants by perceiving unpleasant vs. pleasant odors [The Adaptive Force (AF) is based on HIMA, whereby the holding activity is challenged in particular due to the required adaptation to an increasing external load. This must require even more complex neuromuscular control processes than isometric actions without adaptation to varying external forces. By executing AF, the muscular length-tension control must work properly to maintain stability (isometric position) during the external force increase. If the muscle starts to lengthen during this force rise, the maximal holding capacity  related to the emotion disgust. Since the previous study concerning the effect of emotional imagery on AF had some methodological limitations , the aim of the present study was to verify those findings by investigating the influence of pleasant and unpleasant emotional imagery on AF in healthy individuals in a revised and improved design including randomization of imagery tasks, single-blinding  and baseline measurements.max is significantly reduced during unpleasant food imagery compared to during pleasant imagery and baseline. (2) The maximal AF (AFmax) shows no significant difference between baseline, pleasant and unpleasant imagery. (3) Oscillations occur on a significantly higher AF level (AFosc) during unpleasant vs. pleasant imagery and baseline, but AFosc during pleasant imagery vs. baseline shows no significant difference. In addition to the group comparisons, a case example of a participant being in an actual stressful situation will be presented. The effect of positive imagery under those circumstances will be exemplified thereby.The following main hypotheses were adopted from the previous study: (1) The AFisoIf the previous findings are positively verified, they might show that the holding capability could be a suitable biomarker to test the immediate effect of emotions on motor output, which is relevant to different fields such as neuromuscular control, neuroscience, psychology, sports and movement sciences and medicine. It might provide, furthermore, a better understanding of pathomechanisms of musculoskeletal complaints related to mental health issues, as will be discussed. A positive verification might result in innovative diagnostic approaches for mental but also particular physical health states.The study was performed in the Neuromechanics Laboratory of the University of Potsdam . The AF of elbow and hip flexors of one side was investigated in healthy females by the manual muscle test (MMT) executed by one experienced female tester  using a handheld device to gauge reactions to pleasant and unpleasant food imagery which provoked a feeling of ‘disgust’ or ‘pleasure’. Food imagery was chosen since it is an easy, harmless and ethically justifiable way to trigger either unpleasant or pleasant emotions under comparable circumstances. It was previously shown that experienced testers  are able to perform the MMT reproducibly , which it-test, two-tailed) on the base of the parameter with the lowest effect size of the previous pilot study (zd = 1.390) [n = 9 . In the anticipation of possible dropouts and due to an assumed lower effect size because of the improved design with a presumably lower bias, n = 12 participants were measured.A priori power analysis (G*Power 3.1.9.7) for group differences (dependent = 1.390)  revealedA total of 12 healthy young women volunteered to participate in the study  .The reliable and valid handheld device (max) is attained. The interaction is stopped by the tester and the force decreases again. The duration of the entire force increase (phase 2 to 4) should be ~4 s  as well as the three most tasty (pleasant) and the three most disgusting (unpleasant) foods. The foods had to be rated on a scale from −4 (most unpleasant) to +4 (most pleasant). Those were the base for food imagery during the MMT and were discussed prior to the measurements to obtain precise instructions for the imagery tasks (see below).The second questionnaire was filled in on-site. The current state of mood and physical well-being were obtained (see above). General health questionnaires, such as the SCL-90 (HTS), were not applied since the investigation did not aim at psychological well-being. Coming from movement sciences, the abovementioned scale seems to be sufficient to obtain an impression of the self-reported mood and well-being for the purposes of this investigation.The queried food preferences were discussed between participant, tester and assistants on the measurement day. The participant described the food experience in as much detail as possible and the tester noted the exact words used. Then, the tester asked for individual memorable experiences of the two most pleasant and two most unpleasant foods, e.g., smells, tastes, associations, etc. The aim was to identify triggering and exact words used by the participant to ensure a well-executed imagery task during the MMT trials. Furthermore, the participant chose the most suitable picture of the respective food on a tablet (pictures were from a common online search engine) provoking pleasure or disgust. Across all participants, the most pleasant foods included brownies, pasta with salmon, strawberries, chocolate, pancakes, mango, ice cream, pizza, curry with rice, sushi, BLT sandwich, lasagne, potatoes with quark, mustard eggs, yeast dumplings or hamburger; the most disgusting foods ranged from meat, fish, blood sausage, beetroot, octopus, offal, bananas, Brussels sprouts, spinach, tomatoes on a sandwich, aspic and licorice to pickled gherkins. This highlights the very individual food choices perceived as pleasant or unpleasant. Overall, the unpleasant foods were rated in 13 cases with −4, in 10 cases with −3 and in one case with −2; the pleasant foods were rated 19 times with +4 and five times with +3. In all cases, the foods were connected to individual experiences. For example, the participant who named “BLT sandwiches” as one of her most pleasant foods (+4) said they reminded her of the positive situation of “being on family vacation in Sweden, sitting in the winter garden and watching the lake” while “eating a BLT sandwich with fresh toast, mayonnaise, lettuce, flavorsome bacon and tomatoes”. She reported this as an “experience of great pleasure”. The same participant chose aspic as her most unpleasant food (−4). She described it as “glibbery with pieces of meat inside, it wobbles in the mouth” and “you don’t know what is inside”. This example underlines the highly individual experiences connected with the food consumptions including the related positive or negative emotions.The starting positions of the MMTs of elbow and hip flexors including the handheld device are illustrated in Prior to the measurement day, the participant received information on the study, the consent form and the access to the first questionnaire via email. On the measurement day, the tester introduced the participant to the procedure and the informed declaration of consent was signed before the second questionnaire was filled in. If mood and physical well-being were rated on the scale as >0, the preliminary MMTs of the elbow and hip flexors were executed on both sides (without handheld device). In case of regular stability, the participant was included, and the muscles of her preferred side were measured. If only one side showed regular stability, this side was used for the following measurements. Subsequently, the AF measurements followed. In total, 16 trials were performed, starting with two baseline measurements for each muscle (elbow and hip flexors). Twelve single-blinded, randomized measurements including the imagery tasks followed: each muscle (elbow and hip flexors) was tested three times (M1–M3) during pleasant and unpleasant imagery. The order of muscles and imagery being tested was randomized in Microsoft Excel (Microsoft 365) prior to the measurements. Double-blinding was not possible, since the participants had to actively imagine the food. To ensure single-blinding, the tester left the room while the participant was instructed to enter the imagery by the second assistant using the words noted earlier. The assistant read the words in a neutral and calm manner repeatedly for ~20 s and held the tablet with the chosen picture of the respective food straight above the participant’s eyes at the height of the hanging rail so that the participant kept a supine position without head rotation. Meanwhile, the other assistant prepared the handheld device and tablet for the new trial and recorded any relevant details such as noises. As soon as the participant successfully imagined the respective food , the tester was informed to enter the laboratory. She had no eye contact, neither with the participant (avoided by the curtain) nor the assistants. The first assistant named the muscle to be tested and confirmed that the handheld device was ready for recording (“ok”) without making eye-contact. Any further verbal interaction was prohibited. The participant was previously instructed to focus on her imagination during the MMT. The whole procedure of one trial lasted ~40 s. Resting periods were ~60 s. After each trial, the result of the subjective rating of the MMT (stable/unstable) was given by the tester to the first assistant by a thumb up (stable) or thumb down (unstable) sign without eye contact.After completing all AF measurements, both muscles were tested again for each imagery without blinding, without the curtain and without the handheld device. This checked the tester’s evaluation of the MMTs during pleasant and unpleasant imagery comparing blinded and unblinded trials. Interactions between all persons were allowed again. Subsequently, the participant was asked for feedback. Self-reported information on the imagery process or thoughts during the measurement were of interest and were recorded.TM DIAdem 2021  was used for data processing. The csv-files of the recorded measurements were transferred from the tablet to NITM DIAdem 2021. The force and gyrometer signals were used for evaluation and were first checked visually, which led to the exclusion of trials in some cases.The NISixteen trials were performed per participant . Only 161 trials were evaluated  due to the following reasons: the elbow flexors of two participants and the hip flexors of one participant showed a dysfunction in the preliminary MMT and, therefore, those 24 trials were excluded from evaluation. So as not to change the measuring procedure, they were also measured and not omitted before. Furthermore, one trial of hip flexors had to be excluded because the tester contacted the knee with her chin during the MMT, which might have led to confusion of the tester and/or participant. Visual inspection led to exclusion of further six trials. In one trial a clear pushing by the participant against the tester was visible. This was not allowed since the participants should only perform holding isometric muscle actions. In further five trials the recording stopped before the end of the measurement. It is not clear if the tester pushed the stop button too early or if the measurement stopped because of technical issues.TM DIAdem 2021. The following parameters of interest were extracted for each trial of baseline, pleasant and unpleasant imagery Maximal adaptive force  to gain equidistant time intervals (1000 Hz) and were filtered  in NIogues to ,41):Maximax (N) refers to the maximal value of AF in the force curve that was reached during the entire trial, irrespective of whether the muscle lengthened or not. Thus, it can arise during isometric or eccentric muscle action. If muscle length stayed stable during the whole measurement, AFmax was reached under isometric conditions (AFisomax); if the muscle gave way, AFmax was obtained during eccentric muscle action (AFeccmax). AFmax does not display the participant’s maximal force in general. Under isometric conditions, the AFmax depends also on the force applied by the tester, whereas in case of muscle lengthening, the AFmax is equal to the maximal eccentric force in the present circumstances and is less dependent on the tester’s force application.The AF2.max)Maximal isometric adaptive force (AFisomax (N) refers to the maximal AF, which was reached under isometric conditions. In case of muscular stability, AFmax = AFisomax. In case of instability, it marks the breaking point in which the participant’s muscle merges from isometric to eccentric action. To identify AFisomax, the gyrometer signal (deviation of angle over time) was analyzed. It oscillates around zero under isometric conditions but increases above zero as the muscle starts to lengthen. If the gyrometer signal increased above zero, the force value at the moment of last zero crossing of the gyrometer signal was referred to as AFisomax (breaking point). If the gyrometer signal oscillated around zero throughout the entire force increase, AFisomax = AFmax.This parameter is the most important one for the present investigation. AFisomax was furthermore related to AFmax: max does not necessarily reflect the maximal strength of the participant, a second ratio  and to maxAFmax (%) were calculated, too, to receive information on the force proportion at which oscillations arose regularly. It was previously found that AFosc appeared on a lower level than AFisomax, thus, on a lower level than AFisomax in stable and on a higher level than AFisomax, in unstable MMTs. Therefore, the ratio During the muscular interaction, an oscillating force equilibrium arises, especially in case of muscular stability. It is accompanied by emerging oscillations in the force signal, indicating a clearly distinguishable regular oscillatory behavior (swing up). It was assumed that this might be a prerequisite for muscular stability during the MMT ,41. Therd ~10 Hz ,45,47,484.Slope of force risemax values of all as ‘unstable’ assessed trials of the respective muscle of one participant served as reference. The slope of each force curve (stable/unstable) was calculated by the difference quotient including the time and force values at 70% and 100% of this reference value . The decadic logarithm was applied to obtain the logarithmic slope (lg(N/s)) since the force rise was exponential. If the reference value occurred after the linear phase (transition to force plateau), the trial was excluded from slope analysis to avoid distortion. This was the case in 19 of 161 trials.The force increase during MMT might affect the outcome. In particular, a steeper force rise could compromise the participant’s ability to stabilize the limb’s position. Therefore, similar slopes of force increase were a necessary prerequisite to compare the AF parameters between baseline, pleasant and unpleasant food imagery. Hence, the slope (N/s) of force increase was considered. For that, the arithmetic mean of the AFisoFor the subsequent statistical evaluation, the arithmetic means (M), standard deviations (SD) and 95% confidence intervals (CIs) were calculated for each parameter separately per participant, muscle (elbow and hip flexors) and baseline or imagery (pleasant/unpleasant) and were used for statistical comparisons.Green). For post hoc testing, Bonferroni correction was applied . The effect size eta squared (η2) was calculated in IBM SPSS Statistics. For pairwise comparisons, the effect size Cohen’s d was calculated by zd MD for its standard deviation. The effect size was interpreted as small (0.2), moderate (0.5), large (0.80) or very large (1.3) [p values are given by adjp) and for effect size, Pearson’s r was calculated by r = In total, ten complete datasets for elbow flexors and eleven for hip flexors were analyzed comparing baseline, pleasant and unpleasant imagery. Statistical analyses were performed using IBM SPSS Statistics . The normal distribution of each parameter for each muscle in each condition  was checked by Shapiro–Wilk test. In case of normal distribution, repeated measures of ANOVA were performed (RM ANOVA). If sphericity was not given (Mauchly test), the Greenhouse–Geisser correction was chosen (Fge (1.3) ,50. Becage (1.3) ,52, the The single ratings of the tester for each trial and participant are given in The comparison of the MMT ratings of blinded vs. unblinded trials (without handheld device after AF assessment) showed in most cases that the imagery led to the expected MMT outcome . For elbow flexors, all 20 unblinded MMTs were rated accordingly as stable during pleasant imagery (100%). During unpleasant imagery, the elbow flexors of one participant remained stable for one of the two unpleasant food visualizations (5%), which was in contrast to the hypothesis. All other 19 unblinded MMTs during unpleasant imagery were rated as unstable (95%), according to the hypothesis. For hip flexors, 20 of the 22 MMTs showed stability during pleasant imagery (91%), the remaining two were rated as unclear (9%) and pertained to the same participant. During unpleasant imagery, 21 of 22 unblinded MMTs of hip flexors were assessed as unstable (95.5%), according to the hypothesis; one was rated as unclear (4.5%).Four participants reported to have difficulties imagining the food experiences: for unpleasant ones in general, with repetition of the imagery, the alternation between pleasant and unpleasant imagery or because of discrepancies between the assistant’s instructions and own imagination. In all of those four participants, the result of at least one trial was not according to the hypothesis that AF would be stable during pleasant imagery and unstable during unpleasant imagery . One of the participants with the highest number of discrepancies between expected and occurred result of MMTs (50% according to hypothesis) showed insecurities during the test by, e.g., excusing herself if the muscle was not stable. It seems that she was irritated by the outcome of MMTs in the course of the measurements, especially in case of instability. Two further participants had difficulties with the imagery. They also showed deviations from the hypothesis in 3 of 12 MMTs for hip flexors (25%). Only two of the remaining 6 participants who reported no difficulties in imagining the food experience had one trial each involving the elbow flexors which did not show results according to the hypothesis; thus, the MMT results of those six participants without imagery difficulties were according to the hypothesis in 97% of all trials. This might indicate that the trials which were not assessed according to the hypothesis could result from the imagery quality rather than from the tester’s MMT performance.max; elbow: 100 N, hip: 103 N), which was considerably lower than the AFmax of those trials, which were reached during muscle lengthening . Thus, the muscle started to lengthen at 73% and 62%, respectively, of the maximal force capacity. This reflects an inappropriate adaptation. In contrast, the gyrometer signals during baseline (grey) and pleasant imagery (blue) oscillated around zero throughout the entire force increase, reflecting the quasi-isometric muscle state until the maximum was reached . Minor liftoffs were related to the slight muscle suspension during MMT, which were accepted due to the freely moveable limb [max compared to baseline and pleasant imagery (−32% (elbow) and −45% (hip)). However, the force increased further during muscle lengthening. It is notable for this example that even AFmax was slightly lower for unpleasant vs. baseline and pleasant imagery .The gyrometer signals during unpleasant imagery , red shoble limb . Consequmax was reached). Exempted from this was the MMT of hip flexors during pleasant imagery, whereby oscillations rose simultaneously to AFmax. This will be discussed later. The results of this example are supported by the following statistical group comparisons. The single values of all parameters of each trial, muscle and participant are given in The onset of oscillations did not appear as clear as hypothesized on the basis of the previous studies. The AFosc during unpleasant imagery was 138 N and 153 N for elbow and hip flexors, respectively. For baseline and during pleasant imagery, the AFosc amounted to 133 N and 130 N for elbow and 149 N and 190 N for hip flexors, respectively. Nevertheless, for unpleasant imagery the onset of oscillations appeared clearly after the breaking point, thus, during muscle lengthening; whereas for baseline and pleasant imagery, it occurred under isometric conditions (before AFiso2(2) = 0.722, p = 0.697, n = 10; hip: χ2(2) = 0.250, p = 0.882, n = 8, The prerequisite of similar slopes of force rises for elbow and hip flexors is given indicated by a non-significant difference between baseline, pleasant and unpleasant imagery  = 0.683, p = 0.518), reflecting that AFmax, irrespective if reached during isometric or eccentric muscle action, was similar between baseline, pleasant and unpleasant imagery  (max for baseline vs. unpleasant imagery (t(10) = −2.928, adjp = 0.045, zd = 0.883). Differences between baseline vs. pleasant and unpleasant vs. pleasant imagery were non-significant . AFmax during pleasant imagery was ~9.46 ± 18.05% higher and during unpleasant imagery ~14.84 ± 14.03% higher than AFmax of baseline. During unpleasant imagery, AFmax was ~4.13 ± 13.96% higher compared to pleasant imagery.For hip flexors, RM ANOVA was significant  = 4.777, = 0.323) . Pairwismax for unpleasant imagery was always reached during eccentric muscle action, the decisive parameter for comparison was AFisomax, which was reached during isometric action in each condition .Since AFmax was significantly lower during unpleasant vs. pleasant imagery and vs. baseline  and for pleasant vs. unpleasant imagery . Baseline vs. pleasant imagery differences were non-significant .For elbow and hip flexors AFisobaseline . No signectively c,f. Consmax) was on average 202.43 ± 31.31 N for elbow (n = 10) and 216.82 ± 25.67 N for hip flexors (n = 11). Taking the individual maxAFmax values  = 28.105, p < 0.00001, η2 = 0.757); similar for hip flexors: p < 0.001, η2 = 0.539). For both muscles, pairwise comparisons showed significantly lower adjp = 0.001, zd = 1.798; hip: t(10) = −3.471, adjp = 0.018, zd = 1.047) as well as for unpleasant imagery vs. baseline (elbow: t(9) = −5.559, adjp = 0.001, zd = 1.758; hip: t(10) = −3.888, adjp = 0.005, zd = 1.172), whereby the difference was non-significant for pleasant imagery vs. baseline (both muscles: adjp = 1.000).The overall maximal value of AF  = −5.457, adjp = 0.001, zd = 1.726). max was reached (averaged over both groups: 78.64 ± 13.62%), but during unpleasant imagery they appeared—if at all—after the breaking point for each participant (217.76 ± 108.72%).For elbow flexors, AFosc was significantly lower for baseline and for pleasant vs. unpleasant imagery , indicatGreen = 5.804, p = 0.029, η2 = 0.367). Although AFosc was highest during unpleasant imagery, pairwise comparison of pleasant vs. unpleasant imagery did not differ significantly (Bonferroni correction: adjp = 0.591). Nevertheless, AFosc was significantly lower for baseline vs. unpleasant imagery (t(10) = −3.665, padj = 0.013, zd = 1.105). The values of the ratio AFosc to AFisomax  = 4.747, p = 0.053).For hip flexors, the results showed a slightly different behavior . RM ANOVAFisomax  reflect AFisomax f. RM ANOOne female participant  initially showed an impaired neuromuscular function in preliminary MMTs. A brief talk revealed she experienced current stress . The initial instability of her muscles could be remedied immediately by positive imagery . AF measurements of elbow and hip flexors were performed using the negative imagery (exam situation) vs. the positive ones (yoga/kickboxing) randomized and single-blinded . For negative imagery, the elbow flexors showed an unstable behavior in 2 of 3 trials; in one trial the MMT was rated as stable despite negative imagery. For positive imagery, the elbow flexors were rated as stable for all trials. For hip flexors, all three trials during negative imagery were unstable, for positive imagery, the first two trials were assessed as stable and the last one as unstable. max) was considerably reduced during negative imagery, which was also the entry state of the participant. By imagining positive experiences, the holding capacity was immediately improved, indicating a regular neuromuscular function. The oscillatory onset in stable MMTs occurred before and for unstable MMTs after AFisomax was reached.TheThe aim of the present study was to investigate the influence of pleasant and unpleasant emotional imagery on different AF parameters of elbow and hip flexors in an improved design including randomization, single-blinding and baseline measurements. Since the force rises (slope) did not differ significantly between baseline, pleasant and unpleasant imagery, the subsequent discussion is based on reproducible force rises and, thus, similar performances of the tester’s force application.It was hypothesized the MMTs would be rated as stable for baseline and pleasant imaginations and as unstable for unpleasant ones. The ratings of blinded MMTs did not differ considerably compared to the non-blinded ones assessed without handheld device after the objectified AF measurements. The missing blinding was the main limitation of the previous study. However, 88% of all 121 blinded MMTs (baseline measurements were excluded) and 95% of all 84 unblinded MMTs were rated according to the hypotheses in this study. Thus, even though the tester did not know which imagery was conducted, the subjective ratings of MMTs supported the hypothesis in the vast majority of trials. On that basis it can be stated that blinding did not lead to a considerably different MMT rating by the tester compared to non-blinding. Some limitations regarding imagery quality (mentioned above) must be considered which possibly led to the deviating results.max) during unpleasant imagery compared to pleasant ones and baseline measurements. This result indicates that unpleasant imagery resulted in a worse adaptation capability in healthy participants, irrespective of the muscle. Under the assumption that unpleasant food imagery is related to the negative emotion disgust, this suggests that such negative emotions seem to affect the muscle function in the sense of isometric AF also in healthy participants. The value of this finding will be discussed later.The main hypothesis of this study was supported by the results: the muscles started to give way at a clearly and significantly lower force , the lower AFmax was due to the lower maximally applied force by the tester. Therefore, the adaptive capability of the participant was not challenged by higher forces. However, one outlier existed which might have led to the significant result: the baseline AFmax of one participant was clearly lower compared to the respective AFmax during unpleasant imagery (116.66 N vs. 193.98 N). By excluding this participant, baseline vs. unpleasant imagery did not differ significantly (adjp = 0.096). As mentioned in the methods, the tester stopped the force increase if an oscillatory equilibrium between tester and participant was reached on a considerably high force level. Looking at the onset of oscillations for the baseline trials of this participant, oscillations had already appeared at 58.02 N, which was the lowest of all participants. Those oscillations might have caused a feeling of stability which, in turn, led to termination of force increase by the tester. The essential result was, however, that the AF under isometric conditions was significantly lower for unpleasant imagery, despite of AFmax.The second hypothesis was mainly confirmed by the results. The AFadjp = 0.079). Nevertheless, three participants showed a low AFosc also during unpleasant imagery. It was visible, thereby, that the oscillations were not as clear and as constant as during pleasant imagery and at baseline. This might highlight some methodological limitations for AFosc evaluation.Regarding the third hypothesis (onset of oscillations), the results of AFosc of elbow flexors confirmed the first study, since oscillations appeared at a significantly higher AF during unpleasant vs. pleasant imagery and baseline; furthermore, during unpleasant imagery they appeared—if at all—after the breaking point for each participant, thus during muscle lengthening. For hip flexors, a deviating behavior was found for some participants. A trend according to the hypothesis was visible, e.g., AFosc for baseline was significantly lower than for unpleasant imagery. During pleasant vs. unpleasant imagery, AFosc differed insignificantly, however, the results were close to significance  was adopted from the previous study and was based on the criterion that four consecutive maxima in force increases with a time interval dAnother limitation might be that the quality of imagery was not assessed quantitatively. The self-reporting after the measurements gave an impression of how well the participant interpreted their ability to imagine the food experience. Some participants had difficulties imagining the food, as mentioned above. This might have led to deviations regarding the stability of AF. However, the aim was not to quantify the quality of imagination but their effect on AF in healthy individuals in general. Therefore, they were included.max for baseline measurements vs. unpleasant imagery trials in this study (discussed above), which was not expected. This is presumably a result of the lower maximal force applied by the tester, probably because of perceiving the mutual oscillations already at a low force level. Since the crucial parameter was the AF under isometric conditions (AFisomax), which was still clearly and significantly lower during unpleasant imagery, the lower AFmax of hip flexors for baseline can be neglected here.Possible limitations concerning MMT performance were previously described ,41, espeA detailed proposed explanation of neurophysiological processing during the execution of AF as well as the effect of emotions were given previously ,41,43. Imax immediately. Based on the findings and the practical experience, we propose AF assessment could be suitable to evaluate the positive and negative effects of emotions. Moreover, it seems to be appropriate to test which imagery can improve the holding capacity since this particular muscular function can instantaneously change. It can only be assumed that other bodily systems—autonomous nervous, endocrine and immune systems—behave similarly and would switch from a dysfunction to normal regulation. The underlying potential of such an approach can be imagined thereby and might have applications in rehabilitative and clinical settings. Since not only emotional states are affecting motor processing but also various afferences such as, e.g., nociception [Since several central structures are involved in processing motor control as well as emotions ,7,8,9,10iception ,56, it iIn daily activities and sports, the adaptive capacity of the neuromuscular system is necessary for joint stabilization. If it is reduced, joints could suffer from inappropriate joint alignment under strain, which might result in pain and probably leads to degeneration or increased risk of injury. This could explain the still poorly understood “overuse” injuries and might clarify the causal chain regarding the joint appearance of musculoskeletal pain, muscle weakness and mental stress. From our point of view, mental issues lead to an impaired neuromuscular function which, in turn, can result in complaints of the musculoskeletal system.max ≈ AFmax (≥ 99% of AFmax), indicating the muscle length stays quasi-isometric during the entire force increase. Unstable adaptations, in turn, show a significantly lower AFisomax ≈ 56% of AFmax, reflecting that the muscle starts to lengthen at a significantly lower force during adaptation. Furthermore, during stable adaptation oscillations occur at a force level of ~74% of AFmax; in turn, for unstable adaptation they appear at ~88% of AFmax. In the previous studies and for elbow flexors in the present study, the onset of oscillations for unstable adaptation was at a higher force level (~95%). It is not clear why the oscillations under unstable conditions for hip flexors arose at a low force level (~75%) in the present study. As mentioned, the evaluation or possibly the participant’s regulatory state might play a role. However, it seems to be more important if oscillations arise before or after the breaking point  the oscillations arose after the breaking point, thus, during muscle lengthening. The occurrence of those regular oscillations might be a prerequisite for stability during muscular adaptation. Further investigations are necessary to verify this hypothesis.In the previous studies investigating the AF behavior regarding pleasant and unpleasant imagery/odors ,41, concmax) was reduced highly significantly and instantaneously by imagining unpleasant food experiences. The conclusion is that negative emotions, such as disgust, can lead to muscular instability. Assuming this might result in joint destabilization under strain, it could pave the way for explaining the causal chain regarding the link between musculoskeletal pain and mental health states. It is proposed that an impaired holding function due to mental stress could lead to musculoskeletal pain.The present study investigated the motor adaptation in the sense of AF in reaction to emotional imagery in a single-blinded, randomized setting. The results support the previous findings: the maximal holding capacity (AFisoInvestigating the adaptive holding function might be a promising innovative approach to obtain insights into psychomotor states and to support diagnostics of mental health conditions. AF might be used to test instantaneously the effect of emotions on motor function. This immediate change of muscular stability as reaction to positive or negative stimuli might further help to determine purposeful therapies. This might open up innovative and highly beneficial possibilities regarding psychological diagnostic and treatment approaches. Further research is needed to examine this hypothesis.Moreover, the collected data until now suppose that a proper ‘regular’ neuromuscular adaptation might be characterized by oscillations. This provides novel insights into neuromuscular control. Further research is needed to investigate if they could be a prerequisite for reacting and adapting adequately to external forces.


It has generally been accepted that the current indigenous peoples of the Americas are derived from ancestors from northeastern Asia. The latter were believed to have spread into the American continent by the end of the Last Glacial Maximum. In this sense, a joint and in-depth study of the earliest settlement of East Asia and the Americas is required to elucidate these events accurately. The first Americans underwent an adaptation process to the Americas’ vast environmental diversity, mediated by biological and cultural evolution and niche construction, resulting in enormous cultural diversity, a wealth of domesticated species, and extensive landscape modifications. Afterward, in the Late Holocene, the advent of intensive agricultural food production systems, sedentism, and climate change significantly reshaped genetic and cultural diversity across the continent, particularly in the Andes and Amazonia. Furthermore, starting around the end of the 15th century, European colonization resulted in massive extermination of indigenous peoples and extensive admixture. Thus, the present review aims to create a comprehensive picture of the main events involved in the formation of contemporary South American indigenous populations and the dynamics responsible for shaping their genetic diversity by integrating current genetic data with evidence from archeology, linguistics and other disciplines. These newcomers were descended from Northeast Asian peoples, as demonstrated by a vast wealth of evidence gathered over more than a century from multiple fields of science . Indeed,The settlement of the Americas likely took place after the initial influx of human populations into East and Northeast Asia; therefore, the comprehension of the peopling of America requires first a contextualization of the human dispersion in East and Northeast Asia. Northeast Asia was settled by humans before the LGM, as pointed out by the most ancient archeological evidence: the Yana River site with approximately 31,6 ka BP, near the coast of the Arctic Ocean in northeast Russia , and theAs proposed by the Beringian standstill hypothesis, the ANA would have entered a period of relative isolation from other groups before or during their first dispersal to the American continent . This hyet al., 2018b), giving rise to the northern Native Americans (NNA) and southern Native Americans (SNA) (The Ancient Beringians (AB) split from the ANA  between ns (SNA)  Scheib b, givingns (SNA) ; howeveret al., 2009; et al., 2018), albeit, if so, at quite different times. Massive continental glaciers prevented an interior path to the American continent from Beringia during the LGM . NeverthHitherto all indigenous Americans studied, with few exceptions, are exclusively descendants from the NNA and/or SNA branches, in turn all South Americans studied thus far have SNA ancestry, therefore the initial dispersion into South America must have happened only after the SNA and NNA branches diverged, placing an upper time limit on this event  Posth e. Some poFurthermore, the widespread distribution of a specific set of very characteristic projectile points along with a specific set technological artifacts first discovered in Clovis, New Mexico, in the southwestern United States , was thoIt’s important to note that the archeological sites mentioned above are all post-LGM. However, several pre-LGM sites have been reported, suggesting that human arrival in America might have occurred far earlier. The Pedra Furada  and the Considering a post-LGM initial settlement scenario, not considering controversial South American sites dating from the pre-LGM period, the dispersal of the first migrants from North to South America must have been extremely rapid. According to available evidence, the first humans in South America arrived as early as 15-14 ka BP , althougDemographic models of population dispersion reveal that if the first inhabitants of Brazil arrived at 13,8 ka BP departing from the western opening of the Cordilleran glacier  at 17 ka BP, the required dispersion rate would be 4,1 km per year, a value within the range of what is seen for present-day hunter-gatherers . In any et al., 2018b). It is unknown whether the gene flow from UPA into Mixe interferes with the detection of this relative excess allele sharing; however, this is unlikely the case, as it was previously demonstrated that other groups, for which no evidence of this gene flow has been found, exhibit the same pattern as Mixe; id est, some Native American groups also present less shared genetic drift with Australians than other American indigenous communities . At first glance, the population history of South American natives also appears to inevitably reflect a post-LGM initial presence, because their ancestry can be traced back to the SNA lineage, albeit with a significant genetic structure formed most likely inside the Americas. However, a relative excess of genetic affinity with Australasian populations - including groups from Australia, Melanesia, and South Asia - was detected in some contemporary indigenous communities from the Amazon (Karitiana and Suruí) and the Brazilian central plateau (Xavante) . It was This excess affinity with Australasians was modeled as the contribution of another unsampled population, the now-famous “Ypikuéra”  or “Y” population, which suggested a more complex population history than had been anticipated until that point, most likely involving an additional population influx from Beringia into the continent or the existence of a major genetic structure in Native Americans’ ancestors . In any Interestingly, it has been suggested by a recent study that the divergence between the AB, NNA, and SNA groups might have taken place in Asia , which wThereby, the existence of this Australasian signal opens up a myriad of possibilities for the initial peopling of the Americas, at least from a genetic standpoint. Most intriguing, this involves the possibility of a very early human presence on the continent during or even before the LGM, as long suggested by several archeological sites, though given the lack of pre-Clovis human skeletons, there is still significant debate among archeologists over whether the stone tools discovered were man-made or naturally occurring flakes, among other aspects regarding the validity of these peri and pre-LGM sites . NonetheNotably, this possibility of an additional population influx is not new and has previously been hypothesized based on the existence of the so-called “Paleoamerican” cranial morphology, which has been observed in some individuals from the Lapa do Santo site in Brazil as well as other regions such as Baja California in Mexico . In thisTaken together, the current archeological evidence supports that humans were present in the Americas at least 20 ka BP during the peak of the LGM . AlthougConsidering that continental glaciers completely blocked northern North America throughout the LGM period, an early settlement of the Americas requires an alternate pathway. This alternative is provided by the so-called coastal migration theory (CMT) , which pet al., 2018; Finally, some genetic and morphometric analyses of Northeastern and Southeastern Asians  provide some intriguing pieces of evidence on the origins of the Native American-Australasian connection. First, a link between the Onge and the ancient SEA hunter-gatherers, known as Hòabìnhians, is demonstrated by the genetic affinity between the Onge and two Hòabìnhians from Laos and Malaysia with approximately 8 and 4 ka BP , implyinIn South America, current data suggests that at least three distinct SNA populations dispersed into the continent . The firSouth America, just like North America, according to the available evidence, was settled by very rapid population dispersions along the coast, but this time it most likely occurred along both the Pacific and Atlantic coastlines . In thisAnother hypothesis argues that the Andes, Amazon, and coastal areas were colonized by three separate lineages that split before entering South America . The relationship between the Sambaqui mound builders and the contemporary indigenous communities remains largely unknown due to the relatively small number of individual analyzed until now, however current evidence suggests a higher affinity between them and present-day Jê-speakers, in comparison to other eastern South American natives , ranging from 13 to 2 ka BP, this demographic history is divided into two main phases with distinct demographic dynamics . During Whereas, the spread of sedentary lifestyle and intensification of food production consolidating around 5.5 ka BP, initiated a new period of exponential population growth, at least in some cultural centers, particularly those located in the central and northern Andes. According to this model, more than half of the population growth occurred during this second stage , althougThis shift in human population growth rates also overlaps with a change in the climatic pattern that happened throughout the Middle Holocene, where the climate that was formerly dry and variable in precipitation entered a phase of consistent precipitation increase in the Southern Hemisphere’s tropical forests . If, on This transition to wetter climates is also associated with an increase in sedentism as well as in the prominence of agriculture as a subsistence strategy , as indiSome studies suggest that this exponential rate of growth was maintained until the arrival of Europeans, at least in Amazonia , while oIn addition, the contrast between western and eastern population growth regimes, among other factors, prompted the development of a useful model known as the Andes-Amazon divide , which wet al., 2015), and also that Amazonia was a major world center of crop domestication, with at least 83 species having been domesticated to some extent . These processes were also associated with extensive environmental modifications, such as the formation of domesticated landscapes, exemplified by the Amazonian dark earths (ADEs), which first appeared around 6 ka BP during the mid-Holocene, and became widely distributed by 2.5 ka BP, and were crucial for both plant domestication and food production, and thus for the increased population growth rates  species and leads to coevolutionary dynamics between human genes and culture . In the et al., 2022), both likely tracing back to the initial population events and the serial population bottlenecks faced by these groups, because the effect of genetic drift in small and isolated groups is stronger, resulting in an increased rate of genetic diversity loss; for this reason the Amazon may be home to the world’s living populations with the lowest levels of genetic diversity . Furthermore, isolation by distance also likely plays a significant role in shaping these gradients of genetic variation . Concurrently, genetic divergence among groups - measured by statistics such as Fst - tends to increase from north to south in the Americas and from west to east in South America for the same reasons .Despite the enormous environmental and cultural diversity of South America and Amazonia, there is no equivalency in terms of genetic diversity levels, which are exceedingly low when compared to populations on other continents . This loAs in other regions of the world, crop and landscape domestication, sedentarization, and intensification of food production led to higher rates of population growth in some cultural centers of South America, which in turn caused an increase in human population movement . This prContinental-scale genetic studies of American indigenous populations have not found any significant relationship between genetic and cultural diversity , althouget al., 2019) inferred to have originated on the Pacific coast (Moreover, cultural variables relating to subsistence strategies are likely to be the primary determinants affecting the broader patterns of genetic structure, particularly after the enormous population dispersals of agriculturalist and herder peoples during the Holocene . Therebyic coast , in suchic coast . Eastern South America is no exception, as populations with a predominance of agriculturalist subsistence strategies expanded out of the Amazon. In this sense, Tupi-speaking groups present a pattern of isolation by distance consistent with a past population expansion, in line with the expectations of the Tupi Expansion hypothesis, whereas groups speaking Jê languages and primarily hunter-gatherers show a non-linear pattern of dispersion, which contradicts the expectation of past demic expansions . AlthougSome of South America’s largest language families originated and spread from the Amazon. Furthermore, an intriguing relationship has long been proposed between the geographic distribution of some language families and some of the most important Late Holocene material culture traditions ; while tConversely, the languages of the Arawak and Tupi-Guarani families extended over the territories where their respective material culture traditions thrived and were spoken by local peoples until the European conquest . Indeed,The Tupi-Guarani family and the Tupi expansion are quite emblematic and revealing of the implications of Late Holocene demic expansions to the South American genetic landscape. Currently it is known that all families of the Tupi stock had a common center of origin, most likely located in southwestern Amazonia , betweenCurrent data, including genetic analyses, supports a demic diffusion model along the lines of a long-standing hypothesis that the Tupi-Guarani spread out of Amazonia as a result of ongoing population expansion caused by the emergence of their agriculturalist food production systems . In thisHumans initially arrived in the Americas during or even before the LGM, however the genetic diversity of Native and non-Native American peoples has been greatly altered by the recent events triggered by European arrival. The European colonization of this continent triggered some of the greatest demographic and migration events in human history. At the time of arrival, tens of millions of Native Americans were living on the continent . As wideConcomitantly, there was also widespread miscegenation between these peoples, previously separated by thousands of years of evolutionary history and which now met on the American continent . Thus thThis process of admixture occurred in a differential manner throughout time and space, heavily influenced by the local contexts of indigenous population density, availability of specific resources of interest to Europeans, and the consequent volume of immigrants; but also dependent on other factors such as the intensity of African and indigenous slave labor employment in each specific region, as well as the social and cultural contexts that determined the frequency and volume of immigration and admixture . FurtherNotably, the process of European colonization appears to have had a less severe impact on the indigenous people of the Andes, with slightly smaller population declines and admixture . On one et al., 2019; Recent research has produced a more detailed picture of the genetic structure of South American indigenous peoples, demonstrating a pattern of genetic-geographic relationship . First iFurthermore, indigenous communities in the Southern Cone are genetically distinct from other South American populations, to the point that Central-Southern Chile and Patagonia have an almost unique genetic component, which also has a long-term continuity in the region . FurtherIn general this broad scale population structure of South America was largely in place by the onset of the Late Holocene, resulting in patterns of long-standing genetic continuity , specialThe Amazon is the other major center of origin of population expansions in South America, where numerous indigenous communities were able to flourish despite quite different subsistence strategies and environmental challenges, and eventually some of them experienced substantial population growth and dispersed throughout the continent. The Arawak and Tupi expansions were most likely the biggest in terms of scope and impact, and their genetic imprint may still be seen in indigenous populations in eastern South America and the Caribbean today (Castro e In conclusion, research on native and admixed American populations has gradually revealed the enormous diversity of ancestral lineages for which the Americas represented a meeting point, some of which were separated by tens of thousands of years, such as European and African, brought with colonization and the Atlantic slave trade, respectively. Other lineages, such as those that split during the earliest settlement of Northeast Asia, Siberia, Beringia, and their journey to the Americas, were separated for a shorter period of time, but were nevertheless separated thousands of years ago, towards the end of the Pleistocene. In addition other ancient populations, not sampled thus far, contributed lineages that likely emerged earlier, such as the Australasian lineages, which presumably diverged during the settlement of Southeast Asia. Finally, population dynamics and dispersions, particularly in the late Holocene, caused by climate change as well as the gradual transition to sedentism and more intensive agricultural food production, significantly reconfigured the patterns of indigenous American genetic diversity through inter and intra continental population movements.


C. elegans, HSF-1 mediated heat shock response (HSR) declines sharply on the first day of adulthood, and HSF-1 was proposed to function primarily during larval stages for lifespan assurance based on studies using RNAi. The tissue requirement for HSF-1 in lifespan, however, is not well understood. Using the auxin-inducible degron (AID) system, we manage to uncouple the roles of HSF-1 in development and longevity. In wild-type animals, we find HSF-1 is required during the whole self-reproductive period for lifespan. This period is extended in long-lived animals that have arrested germline stem cells (GSC) or reduced insulin/IGF-1 signaling (IIS). While depletion of HSF-1 from any major somatic tissues during development results in severe defects, HSF-1 primarily functions in the intestine and likely neural system of adults to support lifespan. Finally, by combining AID and genome-wide transcriptional analyses, we find HSF-1 directly activates the transcription of constitutively-expressed chaperone and co-chaperone genes among others in early adulthood, which underlies its roles in longevity assurance.HSF-1 is a key regulator of cellular proteotoxic stress response and is required for animal lifespan. In  Drosophila and mammalian cells, HSF1 activates the HSR by releasing paused RNA Polymerase II (Pol II) at promoter-proximal regions into productive elongation . Upon proteotoxic stress such as heat shock, HSF1 induces the expression of genes encoding molecular chaperones, detoxification enzymes, and protein degradation machinery to cope with stress-associated protein damage and misfolding . It is wongation . Accumulongation . These pongation .C. elegans, Drosophila, and mammals  system to enable rapid depletion of HSF-1 post-larval development in a tissue-specific manner. We also combined HSF-1 depletion by AID with RNA-seq and ChIP-seq analyses to determine the transcriptional program of HSF-1 that underlies its roles in longevity.C. elegans lifespan  <arrested GSC> and daf-2(e1370) <reduced IIS > mutants. As a control for glp-1(e2141), we included another temperature-sensitive mutant fem-3(q20) that is sterile as glp-1(e2141) at the restricted temperature of 25°C < only producing sperm but not oocytes > but has a normal lifespan in the analyses. Similar to wild-type, HSF-1 is required in early adulthood (to at least Day 4 at 25°C) for the lifespan of fem-3(q20), and pan-somatic depletion of HSF-1 since Day 1 of adulthood shortened lifespan by about one third (glp-1(e2141) lifespan, and its functional period is extended in glp-1(e2141) (glp-1(e2141) delays later deaths with an atrophied pharynx but not earlier deaths from pharyngeal pathology by a bacterial infection (fem-3 (q20) and glp-1(e2141) animals on Day 4 (fem-3(q20) are not simply an artifact of defective AID during aging but rather suggest that HSF-1 is less active or its function is minimally required for the lifespan of fem-3(q20) beyond Day 4 at 25°C. While the signal of GSC arrest in glp-1(e2141) more than doubled the lifespan in the presence of HSF-1 (when fed with antibiotic-treated bacteria) (HSF-1 is required for lifespan extension in multiple longevity pathways including those mediated by arrested germline stem cells (GSC) and reduced insulin/IGF-1 signaling (IIS). To understand the roles of HSF-1 in longevity pathways post-larval development, we performed lifespan analyses with HSF-1 depleted at different time points of adulthood in the long-lived ne third ; Table 2nfection . In thison Day 4 , therefodaf-2(e1370) mutant. Due to the high incidence of internal hatching upon HSF-1 depletion on Day 1  and Day 3  of daf-2(e1370), it is difficult to estimate the total contribution of HSF-1 to lifespan throughout adulthood. However, similar to the glp-1(e2141) mutant, daf-2(e1370) also seems to extend the functional period of HSF-1 as depletion of HSF-1 since Day 5 and Day 7 resulted in ∼35% and ∼20% of lifespan reduction in daf-2(e1370) (daf-2(e1370) animals were fed with antibiotic-treated bacteria, in which HSF-1 contributes to longevity up to Day 19 of adulthood   Trial #42(e1370) . This bedulthood  Trial #5C. elegans larval development (Y94H6A.10) or shared by the soma and germline (e.g. hsp-1/hsc-70)  . This re/hsc-70) , HSF-1 cvia RNA-seq analyses following HSF-1 depletion from somatic cells by AID. We performed RNA-seq in the control strains that express TIR1 but have no degron insertion in a time course of auxin treatment (vs. auxin treatment in the control strain) or by degron insertion at HSF-1 (Y94H6A.10 and hsp-1/hsc-70 upon HSF-1 depletion in the soma (bath-36 in this group) lack a correlation between mRNA (24 h post HSF-1 depletion) and Pol II occupancy changes (2 h of HSF-1 depletion), suggesting they are indirectly impacted by HSF-1 . To control for the potential interference by mRNA from the germline and increase the specificity and sensitivity of RNA-seq analyses, we also performed the experiments in the germline deficient glp-1(e2141) background. Indeed, we have uncovered a bigger group of HSF-1 direct targets (28 genes), each of which is associated with one of the 79 HSF-1 binding peaks at the promoter in the soma and decreases mRNA upon 8 h and/or 24 h of HSF-1 depletion , which also supports that they are the primary targets of HSF-1 in adult somatic cells. 19 out of 22 HSF-1 directly regulated chaperone/co-chaperone genes already decrease expression at 8 h of HSF-1 depletion  and F19B2.5/HLTF (encodes an SWI/SNF chromatin remodeler family protein) is activated by HSF-1 in larval development, suggesting that at least part of HSF-1 developmental transcription program sustains in adulthood.We also identified a few nonchaperone genes as HSF-1 direct targets . Especiaelopment , and exp by HSF1 . Similarglp-1(e2141) is unique to this longevity model and how much it is linked to the higher growth temperature (25 vs. 20°C for wild-type), we performed RNA-seq analyses in the fem-3(q20) control at 25°C. The fem-3(q20) shows a similar decrease of HSF-1 direct targets including constitutively expressed chaperones, stress-inducible chaperones as well as nonchaperone genes upon 24-h HSF-1 depletion (glp-1(e2141) compared to that in fem-3(q20) (glp-1(e2141) as one may expect if HSF-1 is hyperactivated by GSC-mediated longevity signal (hsp70 and hsp-16.2 are at higher levels in glp-1(e2141) compared to fem-3(q20), suggesting expression levels of these canonical HSR genes are not solely dictated by the growth temperature. On the contrary, higher mRNA levels of constitutively expressed chaperones are observed in fem-3(q20). This expression pattern is likely linked to the different cell compositions in these two types of animals. Though fem-3(q20) is sterile at 25°C, it still has a germline that makes sperm. The smaller changes upon depletion of HSF-1 from the soma in fem-3(q20) could be well explained by the ‘masking effect’ from unchanged mRNAs in the germline. Consistent with this idea, our recent paper shows that hsc-70 and hsp-90 mRNAs are enriched in the germline while HSR is more robust in the soma (fem-3(q20) with more germ cells shows higher levels of constitutive chaperones but lower levels of inducible chaperones in whole animal analyses. Collectively, the HSF-1 transcriptional program that we identified in the germline deficient glp-1(e2141) likely applies to somatic cells of animals with normal lifespan as seen in fem-3(q20). Future studies are needed to determine whether HSF-1 activities are enhanced by longevity signals in the absence of external stress.To test whether the transcriptional program of HSF-1 identified in epletion . Althougm-3(q20) , not ally signal . The mRNthe soma , therefoWhile most of the HSF-1-dependent, constitutively-expressed chaperones and co-chaperones have roles in all cell types, over-expression of HSF-1 in specific tissues  is sufficient to extend lifespan . To undeWe then tested the tissue requirements of HSF-1 for larval development. Loss of HSF-1 in any of these tissues since egg lay led to larval developmental arrest or delay . HSF-1 dglp-1(e2141) as the model since pan-somatic depletion of HSF-1 in this background exhibited the biggest lifespan shortening effect, therefore, providing sufficient dynamic range to examine potentially smaller effects from HSF-1 depletion in a single tissue type. Depletion of HSF-1 in the intestine and neural cells both resulted in ∼55% lifespan shortening  and the control fem-3(q20) to compare HSF-1 activities is inconclusive due to the different cell compositions of these animals. Future work with tissue-specific transcription measurement is needed to test whether longevity signaling  hyperactivates HSF-1 in physiological condition (not upon stress) and/or sustains HSF-1 activities longer during aging. Consistent with the idea that functions of HSF-1 in development and longevity can be uncoupled, we found the tissue requirements for HSF-1 in larval development and lifespan are different  system to enable rapid and efficient depletion of HSF-1 and determined the spatiotemporal requirement for HSF-1 in lifespan post-larval development. We found that HSF-1 is predominantly required during the early adulthood to support lifespan, which overlaps with the self-reproductive period in wild-type animals ; Table 1ifferent . Loss ofDrosophila and mammalian cells, where HSF-1 functions at releasing promoter-proximally paused Pol II into productive elongation  or causes Pol II accumulation at the promoters (e.g. hsp-110) during heat shock  genes induced by auxin. This gene list  not onlyongation . It is aat shock . The diftor NELF .pat-10 promoter or depletion of HSF-1 alters its expression, implicating regulation of PAT-10 is likely unique to HSF-1 overexpression. HSF-1 directly activates a compact transcriptional program including classical stress-inducible chaperones, constitutively-expressed chaperones, and co-chaperones as well as a few nonchaperone genes  and fem-3(q20) animals were maintained at 15°C and grown at 25°C (since L1) for experiments.Unless stated, chniques . The temhsf-1 gene as detailed in our previous publication (eft-3p:tir1:mRuby) and swapping the eft-3 promoter with tissue-specific promoters. We first removed the eft-3 promoter through microinjection of two chemically modified synthetic sgRNA (Synthego) in CA1200, which are against the upstream and downstream regions of the eft-3 promoter, along with Cas9 Nuclease  following the previously published protocol (rab-3b (1.2 kb), dpy-7 (350 bp), and myo-3 (2.5 kb) promoters were used for the expression of TIR1 in neural cells, hypodermis, and muscle. The published allele in CA1209 (ges-1p:tir1:mRuby) was used for intestinal expression of TIR1. All the transgenic models made by CRISPR were outcrossed 6 times before use.The HSF-1 AID models were made by CRISPR knock-in of aid:gfp to the C-terminus of endogenous lication . New tisprotocol . The DNAAuxin treatment was performed by transferring worms to bacteria-seeded NGM plates containing 1 mM (if not specified) or 0.5 mM auxin . The preparation of auxin stock solution (400 mM in ethanol) and auxin-containing NGM plates was performed as previously described . In all The HSF-1 AID animals and the corresponding control animals that only express TIR1 were age-synchronized by egg lay for 1 h on EtOH or Auxin plates. Larvae were grown for the indicated time  and crawFor brood size analyses , animalsAge-synchronized worms were scored as dead and removed in the absence of touch response or pharyngeal pumping 6 days per week. For fertile animals, worms were transferred to fresh plates every day through the reproductive period to remove progeny. Bagged, desiccated, or missing animals were censored from analysis.E. coli OP50 cells were spun at 3000 × g for 20 min and resuspended in the same volume of M9 buffer supplemented with 0.5 mM of carbenicillin. The bacteria were then incubated in the shaker at 37°C for 3 h. The bacteria were concentrated by 5 fold  and seeded onto 10 cm NGM plates using 400 ul of concentrated bacteria per plate.In a subset of lifespan experiments, carbenicillin treatment of OP50 was performed as previously described to prevent bacterial growth . BrieflyImaging of live animals was done by immobilizing age-synchronized young adult animals in a drop of M9 buffer containing 6 mM levamisole on a 2% agarose pad. Fluorescent images  were acqglp-1 (e2141) and fem-3(q20) backgrounds, synchronized L1 larvae were grown for 48 h at 25°C on 10 cm normal NGM plates (∼500 worms per plate) to develop into young adults. Approximately 120 young adult worms were picked onto 10 cm NGM plates containing either EtOH or auxin and kept for an indicated time before collection . For each condition, RNA was extracted using a 300 μL Trizol reagent. Worms were vortexed continuously for 20 min at 4°C and then went through one cycle of freeze-thaw to help release RNA. Following this, RNA was purified using the Direct-zol RNA MiniPrep kit (Zymo Research) as per the manufacturer’s instructions using on column DNase I digestion to remove genomic DNA.The HSF-1 AID animals and the corresponding control animals that only express TIR1 were synchronized by treatment of alkaline hypochlorite solution (bleach). The experiments in JTL611, JTL621 (HSF-1 AID models), and CA1200, CA1199 (control) were done as described in our recent paper . For theTotal RNAs were polyA enriched, and directional RNA-seq libraries were prepared using the NEBNext Ultra II RNA library prep Kit. Paired-end sequencing was done at a NovaSeq 6,000 sequencer at OMRF clinical genomics core. The majority of samples were sequenced by 50 bp. A subset of samples that were sequenced with longer reads was trimmed to 50 bp to make all downstream mapping and analyses consistent.http://david.abcc.ncifcrf.gov/) with functional annotation clustering to collapse redundant GO terms. The enrichment score for each cluster was shown.RNA-seq analyses were conducted as previously described . BrieflyChIP-seq reads mapping, peak calling, and generation of genomic occupancy files (bedgraph files) were detailed in our recent publication . To assiglp-1; eft-3p:tir1; hsf-1:degron) were synchronized by bleach synchronization, and L1 larvae were grown on NGM plates containing ethanol at 25°C for 48 h. Young adult worms were rinsed off in M9 buffer and plated on either ethanol or auxin plates for 48 h before rinsing for freeze-cracking. A methanol incubation for 15 min at 4°C after fixation was added to eliminate the TIR1:RFP signal in the worms. The samples were co-stained with Anti-Ubiquitinylated proteins  at 1:200 and anti-REC-8  at 1:100 at 15°C overnight. We then labeled FK2 with anti-mouse-Alexa 647 (Invitrogen Cat# A32728) and anti-REC-8 with goat anti-rabbit-Alexa 488 (Invitrogen Cat# A32731) by incubating for 2 h at room temperature.Worms were prepared by freeze-cracking and fixed in 4% paraformaldehyde, as described  with thehttps://sbi.postech.ac.kr/oasis2/). RNA-seq and ChIP-seq analyses are described in Materials And Methods.Statistical significance was calculated by unpaired, two-tailed Student’s t test in Microsoft excel or two-way ANOVA comparison in GraphPad Prism. Lifespan statistics were calculated by Log-rank test using OASIS2 online lifespan analysis software (


Eranthis, an early flowering perennial herb, can serve as a good model to study genetic differentiation and gene flow due to its easily detectable population characteristics and known reproductive strategies, which can be associated with gene flow patterns. Eranthis populations are typically small and geographically separated from the others. Moreover, previous studies and our own observations suggest that seed and pollen dispersal between Eranthis populations is highly unlikely and therefore, currently, gene flow may not be probable in this genus. Based on these premises, we hypothesized that the genetic differentiation between the Eranthis populations would be significant, and that the genetic differentiation would not sensitively reflect geographic distance in the absence of gene flow. To test these hypotheses, genetic differentiation, genetic distance, isolation by distance, historical gene flow, and bottlenecks were analyzed in four species of this genus. Genetic differentiation was significantly high, and in many cases, extremely high. Moreover, genetic differentiation and geographic distance were positively correlated in most cases. We provide possible explanations for these observations. First, we suggest that the combination of the marker type used in our study (chloroplast microsatellites), genetic drift, and possibly selection might have resulted in the extremely high genetic differentiation observed herein. Additionally, we provide the possibility that genetic distance reflects geographic distance through historical gene flow, or adaptation in the absence of historical gene flow. Nevertheless, our explanations can be more rigorously examined and further refined through additional observations and various population genetic analyses. In particular, we suggest that other accessible populations of the genus Eranthis should be included in future studies to better characterize the intriguing population dynamics of this genus.Genetic differentiation between populations is determined by various factors, including gene flow, selection, mutation, and genetic drift. Among these, gene flow is known to counter genetic differentiation. The genus  In our study, under the low probability of gene flow, genetic differentiation between populations was significantly high and genetic distance generally reflected geographic distance in the genus Eranthis. We suggest that significantly high level of genetic differentiation is due to the marker type used, genetic drift, and selection. We also suggest that the positive correlation between genetic distance and geographic distance might have resulted from historical gene flow or adaptation without historical gene flow. In addition, it has been reported that populations are often separated by forest roads or mountain trails within the same site .In the cases of Eranthis cannot disperse over long distances, and from personal observation, the dispersal distance would be only up to 1 m. The species in this genus produce rounded or sub‐globose seeds approximately 2–3 mm in diameter  may not disperse long distances.Meanwhile, some reports indicate that the seeds of the genus E. byunsanensis and Megaleranthis saniculifolia, the latter of which is morphologically and taxonomically similar to the genus Eranthis, resulting in low possibility of long‐distance pollen dispersal . Dried leaves or bracts of about 20 mg were crushed with Tissue Lyser (QIAGEN), and DNA extraction was conducted using RNase A and the buffers in the DNeasy Plant Mini kit. The extracted DNA was then diluted to a final concentration of 10–50 ng/µl for the downstream analyses.The collected leaves or bracts were dried with silica gel and voucher specimens were deposited in CBNU, the herbarium of Chungbuk National University in South Korea. DNA was extracted from 935 individuals of the four 2.2Eranthis species. These loci were randomly selected from 24 cpSSR loci isolated by Oh and Oh (Twelve chloroplast microsatellite (cpSSR) loci  were genotyped in the 935 individuals of the four h and Oh .PCR was conducted in a TAKARA PCR Thermal Cycler Dice Touch (TAKARA Bio Inc.) using the Multiplex Master Mix of the QIAGEN Multiplex Master kit (QIAGEN). The PCR amplification conditions and procedures were the same as those described by Oh and Oh .An ABI3730xl DNA Analyzer (Applied Biosystems) and GeneMapper v. 3.7 (Applied Biosystems) were then used to measure PCR product lengths.Eranthis were used to analyze genetic diversity, population structure, and other evolutionary parameters.In the previous phylogeographic study of the authors Oh & Oh, , the sam2.32.3.1Eranthis species, genetic differentiation between populations was estimated using two parameters, FST , is less sensitive to high mutation rates reported in microsatellite, thus complementing the weakness of the FST  4.05 n et al. , was use2.3.3ST in this study) and geographic distance. In other words, this test was conducted to identify whether we can find the pattern of isolation by distance in the data. The Mantel tests were conducted using GenAlEX 6.5 . This measure was taken because analyzing all 20 populations in a single run would have been extremely time‐consuming, and even if the analysis itself was completed, there was a possibility that the results would not have been meaningful .The coalescence‐based MIGRATE‐N 3.6.11  and mutation‐scaled immigration rates  were determined. The number of immigrant individuals per generation was calculated by multiplying θ by M. Bayesian inference was used in these analyses and the Brownian motion model was selected as the data type. The results of applying a Brownian motion model are very similar to those of applying a stepwise mutation model, but the time it takes to analyze is much shorter with the Brownian motion model. Other parameters in the MIGRATE‐N analysis were set to the default values or were established based on the suggestions provided by the MIGRATE Manual V. 3.2.1 Beerli, .The relationship between migration rate and geographic distance was analyzed using a regression analysis tool in Excel 2013 to determine if the level of migration was affected by geographic distances between the populations in each species.2.3.5BOTTLENECK 1.2  to 0.93366 (0.93), and RST values ranged from 0.10817 (0.11) to 0.99894 (1)  and all the other populations were extremely high, ranging from 322.9612 to 347.0752 52 Table . The oth52 Table . In the .1 Table . The lowns Table . The higns Table . This reOh & Oh, , where t3.2ST) and geographic distance in the focal species, and the Mantel test , the long‐term migration rate (M), and the number of migrants (Nm) were calculated in five groups (1. E. byunsanensis and E. pungdoensis populations ranged from 1.222 (95% CI: 0.0–3.5) to 16.079  to 79.766 (95% CI: 56.5–98.0), and these values were much higher than those of the other groups  to 46.117 (95% CI: 39.0–54.0), and the number of migrants ranged from 0.444702 to 31.136354 .In 44.1ST and RST were significant in all cases, with the exception of one E. pinnatifida instance , no allozyme diversity was detected, whereas 23 haplotypes were recovered with 9 chloroplast microsatellite markers , and different selective pressures likely acted in the different locations where these populations were located, resulting in different genetic characteristics between the populations. Even though morphological differences are not clearly observed between these widely distributed populations, adaptive genotypes might have still been generated, resulting in high genetic differentiation. However, in our study, selection as an important factor affecting genetic differentiation is not yet supported by actual analyses or observations, and further research is needed to define the extent to which the high genetic differentiation observed is due to selection. Overall, the high genetic differentiation values in our study are likely due to a combination of all of the aforementioned factors, which are the genetic marker used, genetic drift, and possibly selection.Finally, the high genetic differentiation observed herein might have also been due to selection. Selection can genetically structure populations when organisms are selectively adapted to gradient environments, which is also referred to as isolation by environment or isolation by adaptation 2, which indicate that genetic distances are clearly correlated with geographic distances in the absence of current gene flow.As mentioned above, we initially hypothesized that geographic distances between populations would not meaningfully affect genetic distances in the absence of gene flow considering that gene flow would be the major factor which can generate the correlation between geographic distance and genetic distance. However, our results support that the values of FE. byunsanensis and E. pungdoensis, the values of FST, RST, (δµ)2, and the Mantel test results indicate that genetic differentiation does not reflect geographic distance, which appeared to support our initial hypothesis.In the case of E. pinnatifida and E. stellata, a clear correlation was observed between genetic differentiation and geographic distance. For example, in E. pinnatifida, Hiroshima populations PH5, PH8, and PH9, which are located close to the others (10–30 km), exhibited significantly low genetic differentiation (FST and RST) and genetic distance (δµ)2. Similarly, two Shiga populations of this species where the geographic distance was approximately 20 km also exhibited a notably low genetic distance.However, in Eranthis. However, additional studies are required to identify the mechanisms by which genetic differentiation reflects geographic distance in Eranthis populations in cases where gene flow between populations seems to be impossible. Here, we propose two different hypotheses that could explain the counterintuitive correlations between geographic distance and genetic distance (genetic differentiation) observed herein.Overall, as discussed above, genetic differentiation and genetic distance appeared to reflect the geographic distance in many cases within the genus First, it is probable that gene flow historically existed, and this past event determined the current correlation between genetic differentiation and geographic distance. Our historical gene flow analysis using MIGRATE‐N demonstrates that there existed substantial gene flow long time ago, supporting this hypothesis. The occurrence of past gene flow means that geographic distance had previously affected gene flow , soil properties, and other abiotic / biotic conditions. Generally, populations that are separated by long distances tend to inhabit substantially different environments and experience different evolutionary pressures compared to populations in closer proximity. This correlation between geographic distance and environmental difference is well established ; Data curation (lead); Formal analysis (lead); Investigation (lead); Writing – original draft (lead). Byoung‐Un Oh: Conceptualization .The authors declare no conflict of interest.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


The bottlenose dolphins are one of the most used species in entertainment, assisted therapy, education, and research on welfare. However, their maintenance in captivity requires powerful and sensitive tools for preserving their diversity. The number of genetic markers for this purpose remains controversial, restraining the marine species’ genetic diversity determination. We aimed to select 15 hypervariable molecular markers whose statistical parameters were made in 210 captive dolphins from 18 Mexican centers to support their usefulness. The proposed set of markers allowed us to obtain a genetic fingerprint of each dolphin. Additionally, we identified the structure of the captive population, analyzing the groups according to the capture location. Such characterization is key for maintaining the captive species’ biodiversity rates within conservation and reintroduction programs. However, these 15 genetic markers can also be helpful for small- isolated populations, subspecies and other genera of endangered and vulnerable species. Tursiops truncatus using microsatellites, its informative degree has been poorly reported. Furthermore, the genetic structure of this cetacean has not been fully studied. In the present study, we selected 15 microsatellites with which 210 dolphins were genetically characterized using capillary electrophoresis. The genetic assertiveness of this set of hypervariable markers identified one individual in the range of 6.927e13 to 1.806e16, demonstrating its substantial capability in kinship relationships. The genetic structure of these 210 dolphins was also determined regarding the putative capture origin; a genetic stratification (k = 2) was found. An additional dolphin group of undetermined origin was also characterized to challenge the proficiency of our chosen markers. The set of markers proposed herein could be a helpful tool to guarantee the maintenance of the genetic diversity rates in conservation programs both in Tursiops truncatus and across other odontocetes, Mysticeti and several genera of endangered and vulnerable species.Genetic analysis is a conventional way of identifying and monitoring captive and wildlife species. Knowledge of statistical parameters reinforcing their usefulness and effectiveness as powerful tools for preserving diversity is crucial. Although several studies have reported the diversity of cetaceans such as  Tursiops truncatus, Montagu, 1821) is among the most mobile species, presenting a broad distribution in the different oceans and polar coastal waters [Delphinidae  is a diverse family including at least 12 genera and 37 species [l waters .T. truncatus has been characterized depending on its distribution in coastal (>20 m isobath), continental shelf (between 20–200 m depth), inshore , and oceanic (>200 m depth) waters [T. truncatus is one of the most used animals in the recreational industry and assisted therapy, being one of the most widely distributed specimens in captivity [) waters . Each as) waters . Given iaptivity . In addiaptivity ,9,10.Ley General de Vida Silvestre” (article 60 Bis), any species of marine mammal must not be subject to extractive use, whether for subsistence or commercial purposes [In agreement with the Mexican “purposes . Nonethepurposes ,12,13,14purposes . Such inpurposes . Howeverpurposes .Despite its low-cost, morphological identification could be a subjective feature. To promote the most effective protection, the characterization of the genetic population architecture (diversity and structure) is crucial for efficient and effective preservation practices . Thus, tMicrosatellites or short tandem repeats (STRs) are multiallelic markers appropriate for population genetic studies and pedigree analyses. These hypervariable molecular markers may be amplified with modest DNA concentrations, being a cheaper process than next-generation sequencing methods . HoweverT. truncatus) from 18 Mexican dolphinariums were characterized using 15 STRs. This set of markers allowed us to genetically distinguish each dolphin with remarkable efficiency (discrimination power ≥ 0.999), evidencing its robustness at a low cost. Our findings validated using these 15 STRs to evaluate the probability of identity and characterize the genetic structure and diversity of genome banks used in artificial insemination, which has been poorly described.In this study, the genetic diversity and population structure of 210 captive dolphins .The present study was conducted in agreement with the Federal Attorney for Environmental Protection  in Mexico City to carry out joint actions for the diagnosis, surveillance, prevention, and control of diseases in any animal species whose survival is threatened. The study was approved by the Research Committee of the T. truncatus living captive. The capture locality was collected in 177 individuals from expedients, grouping these specimens according to this information. Nevertheless, its demographic history was unknown. Out of 210 dolphins, 104 were putatively assigned to Mexican waters . Likewise, 46 dolphins were assumed to be from Cuban territorial waters (CD), 14 from southern Australian waters (AD), and 5 from Japanese (JD) waters. The rest of the dolphins (41) had an unknown geographic origin   .260/λ280) and concentration (ng/μL) were evaluated with Nano-Drop 1000 . DNA integrity was checked with agarose  gel 0.8% stained with ethidium bromide .Genomic DNA was isolated using the MagNA Pure LC Total Nucleic Acid Isolation kit  in the MagNA Pure LC automated equipment . DNA purity . The amplification conditions consisted of 35 cycles of an initial denaturation at 94 °C for 3 min, followed by a denaturation at 94 °C, annealing . Allelic assignment was performed with the GeneScan v3.2 software .Samples were genotyped with 15 microsatellite reported . These 1k), and expected (He) and observed (Ho) heterozygosity using Arlequin v3.5.2.2;  [F statistics (ISF) was estimated with Gènetix v4.05.2; Gènetix team  using 10,000 permutations. Linkage disequilibrium (LD) was estimated with FSTAT v2.9.3.2;  using 105,000 permutations [The obtained genotypes were subjected to statistical analyses, including allele and genotype frequencies, number of alleles (zerland) . The numzerland) . Hardy–Wutations .locus with Genepop v4.6 and FreeNA and confirmed in Micro-Checker v2.2.3;  [Null alleles and large dropouts were checked for each ull, UK) ,34.Matching probability (MP), discrimination power (PD), and the polymorphic information content (PIC) were assessed using PowerStats v1.2; Promega CORP. .STF values) were assessed with Arlequin v3.5.2.2;   and adjusted by a false discovery rate test in R-software;  [The genetic structure was inferred with Structure software v2.3.3;  using 100,000 burn-in and 700,000 after burn-in interactions . Five in Israel) ,38. Thes Israel) .loci presented at least seven different alleles with a mean PIC of 0.766. The most informative markers were Ttr63 (k = 26), EV37 (k = 24), and MK06 (k = 18), whereas TEXVET07 (k = 7), MK09 (k = 8), Ttr58 (k = 8) and KWM02 (k = 9), were the least informative  with a highly efficient PD (PD ≥ 0.999).MP and PD are summarized in the ISF mean = 0.198; range: 0.058–0.423), we determined and confirmed null alleles using two different programs. The null allele frequencies ranged from 0.023 to 0.185 with wide confidence intervals . The HWD was related to a homozygosis excess (ISF > 0). A significant number of loci pairs exhibited LD adjusting the p-value for 5% (p ≤ 0.00047) and 1% (p ≤ 0.000095) of the nominal level  in 13 out of 15 al level .FST values were also estimated to compare the variation among the populations; UD was included in this analysis . Nevertheless, MD1 and MD2 did not differ significantly regarding UD , efficiently in distinguishing one individual in a range of millions of dolphins. Such effectiveness was comparable with the data obtained from 19 STRs, although more cost-effective [Previous studies have used multiple genetic markers for identification purposes ,27,28,29ffective . These fffective ,27,28,29ISF values found suggest “inbreeding”, impacting the genetic variance and covariances between relatives [loci and molecular assessments to monitor the diversity [Balaena, Balaenoptera, Delphinapterus, Eschrichtius, Globicephala, Grampus, Megaptera, Stenella, Orcinus and Pontoporia, to mention a few [Of note, the probability of identity was obtained assuming random mating. Notably, the elatives . Hence, elatives . Such “ielatives ,44. The elatives . Furtheriversity . Given ton a few ,27,28,46T. truncates is not considered an endangered species, these markers allowed us to determine its genetic structure by identifying two well-defined populations [Tursiops. These studies should also consider the environmental changes that have altered the genetic structure, suggesting that geography is only one of the different aspects contributing to genetic differentiation [Even though ulations . Such stulations ,48,49,50ulations ,49,51,52ulations . Geneticulations . Thus, iulations ,56. Our ulations . Such dintiation .On the other hand, the knowledge of the genetic structure has been associated with the reintroduction facility, being successful where both populations (reintroduced and acceptor) share genetic similarities . DiversiTursiops aduncus (T. aducus) that could have been confused with Tursiops truncatus truncatus (Tursiops t. truncatus). The genetic characterization using STRs and mtDNA has shown the presence of T. aducus in coastal waters of southern Australia [Tursiops t. truncatus and T. aduncus. Nonetheless, not all authors share their raw data or analyze the same loci, hindering such analysis. In this setting, the “inbreeding” exhibited by the studied populations was inconsistent with the highest diversity values and the greatest number of alleles, reinforcing this possibility.Regarding the remarkable diversity found, we did not rule out that it could be related to other species such as ustralia . Unfortuustralia ,49,62. AT. truncatus, their reintroduction is not feasible, mainly because they are not vulnerable populations and are dependent on humans. Foraging strategies and social relationships have been modified in captive dolphins due to human dependence [Although genetic characterization is a key topic in the conservation and reintroduction process, it is only one of the diverse variables that should be considered ,43,63. Vpendence . In suchpendence . Thus, rpendence .As mentioned before, the new regulations  did not prohibit scientific research and education programs  Thus, thIn summary, the results presented herein validated using these 15 STRs to evaluate the probability of identity and to characterize the genetic structure with remarkable efficiency. These hypervariable markers are powerful and sensitive, enabling the genetic characterization of this and other species at a low cost, preserving the diversity amongst individuals. Nevertheless, more studies involving uniparental markers, sex determination, cranial and other morphological measurements should be determined to complete such a panorama.