Although the association of human temperament and preference has been studied previously, few investigations have examined cerebral cortical activation to assess brain dynamics associated with the motivation to engage in performance. The present study adopted a personality and cognitive neuroscience approach to investigate if participation in ballroom dancing is associated with sensation-seeking temperament and elevated cerebral cortical arousal during freely chosen musical recall.Preferred tempo, indicated by tapping speed during melodic recall, and a measure of fundamental disposition or temperament were assessed in 70 ballroom dancers and 71 nondancers. All participants completed a trait personality inventory  to determine four primary types: choleric, sanguine, phlegmatic and melancholic. Participants separately recalled their favorite musical piece and tapped to it with their index finger for 40 beats using a computer keyboard. A subset of 59 participants  also repeated the same tapping task while electroencephalographic (EEG) activity was recorded.The results revealed that the dancers were more extraverted, indicative of a heightened need for arousal, exhibited a preference for faster musical tempo, and exhibited elevated EEG beta power during the musical recall task relative to nondancers. Paradoxically, dancers also showed elevated introversion  relative to nondancers, which can be resolved by consideration of interactional personality theory if one assumes reasonably that dance performance environment is perceived in a stimulating manner.The results are generally consistent with arousal theory, and suggest that ballroom dancers seek elevated stimulation and, thereby, choose to engage with active and energetic rhythmic auditory stimulation, thus providing the nervous system with the requisite stimulation for desired arousal. These results also suggest an underlying predisposition for engagement in ballroom dance and support the gravitational hypothesis, which propose that personality traits and perception lead to the motivation to engage in specific forms of human performance. Many explanations of human behavior and performance consider an individual’s temperament or personality, which implies that there are pre-existing differences that draw people into different activities  . The preWhen music starts, the spontaneous movements of humans will typically synchronize with the auditory stimulation, resulting in a link between tempo and motor behavior. In addition, neural activity is positively associated with the tempo. For example, when a listener is presented with music at a tempo of 2.4 Hz, a corresponding  neural oscillation is present in the electroencephalographic (EEG) activity . Additiocholeric, which corresponds to neurotic extraverts; sanguine, corresponding to stable extraverts; melancholic, consistent with neurotic introverts; and phlegmatic, consistent with stable introverts  and 71 nondancers , all of whom were of Chinese nationality, were recruited from flyers posted in the student lounge describing the study and asking for volunteers. All of the dancers majored in dance sports, had at least 5 years of experience and were right-handed. They had no observable cognitive dysfunction, motor or mental disorder that could affect their ability to perform the task.To achieve a statistical power of 80% to conduct statistical assessments of the psychophysiological measure, a power analysis indicated that 40 participants were required. A subset group of 29 dancers  and 30 nondancers  also were tested with EEG to explore the neural mechanisms of preferred tempo while they completed the tapping task. The study procedures were consistent with the ethical standards of the Research Ethics Committee and with the 1964 Declaration of Helsinki. The experiment was approved by the ethics committee of the Shanghai University of Sport (No. 2015003) and all participants read and signed an informed consent form before the experiment. Participants were compensated for their participation.I’d rather work alone than have a lot of people together (宁可一个人做事，不愿与很多人在一起)”, “I am good at interacting with people (善于和人交往)”, “Act rashly, often without thinking of the consequences (做事有些莽撞，常常不考虑后果)”  to 2 (very consistent), having 15 items associated with each temperament type. For instance, “常不考虑后果)” . The fouTo achieve the purpose, we assessed temperament and preferred tapping tempo during melodic recall in dancers and controls during an initial phase of data collection. To further explore the neural activation, we ran a second phase of data collection, which was identical to the first phase in terms of the temperament assessment, however, EEG was also collected during melodic recall and execution of tapping. As such, we examined how temperament and cerebral cortical activation were related to tempo preference from both behavioral and neural levels of measurement through employment of the second phase of testing.Participants first sat alone in a quiet room and completed a temperament questionnaire. Second, they performed a tapping task for which they were asked to recall their favorite music piece and tap to the tempo using the letter “M” on a computer keyboard for 40 taps. A computer  provided visual instructions and recorded tapping data using routines from Psychtoolbox 3.0 running on MATLAB 2013a . The screen  was placed 80 cm in front of the seated participants. In addition, we also recorded the EEG  across the scalp topography during the recall period in subset group, which was the time taken to complete the 40 taps in the subsample of participants.The EEG was recorded using 64 Ag/AgCl electrodes placed on the scalp according to the international 10–20 system. The ground was placed at AFz and was referenced to the FCz site on-line. Re-referencing was achieved with two electrodes placed on the mastoids in pre-processing. The horizontal electrooculogram was recorded at the outer canthus of the right eye, and the vertical electrooculogram was recorded below the left eye. Impedances of all electrodes were kept under 5 kΩ. EEG was recorded continuously until the participant finished tapping and was digitized at a sampling rate of 1000 Hz .EEG preprocessing was performed with the software Brain Vision Analyzer 2.01 . The EEG data were then analyzed offline using EEGLAB  in MatlaAnalysis of the power spectrum was conducted by performing short-time Fourier transform (STFT)  on the ft-tests were conducted to determine if there was a difference in the preferred tempo and variability between the two groups. Multivariate analysis of variance (MANOVA) and a 2 (Group: dancer vs. nondancer) × 4  ANOVA was applied to the scores of the four temperament types to determine differences between and within the groups. A series of correlational analyses was also conducted to assess the association between preferred tempi and temperament in each group via the Pearson correlation coefficient.The preferred music tempo was calculated as beats per minute (bpm) to represent the rate. The standard deviation (SD) of the beat among the 40 repeated taps was also examined to evaluate the variability of an individual’s preferred tempo. Independent For EEG data, a 2 (Group: dancer vs. nondancer) × 2 (Hemisphere: left vs. right) × 5  ANOVA  and a separate 2 (Group: dancer vs. nondancer) × 4  ANOVA were applied to examine theta (4–8 Hz), low-alpha (8–10 Hz), high-alpha (11–13 Hz), low-beta (13–20 Hz) and high-beta (21–30 Hz) power. Correlation  analyses were also conducted to examine any associations between preferred tempo and brain dynamics .t-tests applied to the preferred tempo revealed that the tempo preferred by dancers (159 ± 65 bpm) was significantly faster than that preferred by nondancers (132 ± 66 bpm), t = −2.427, p = 0.016, Cohen’s d = 0.409, but there was no significant effect for SD, ps > 0.05.Independent F =15.730, p < 0.0001, ηp2 = 0.102 and F = 15.723, p < 0.0001, ηp2 = 0.102, respectively. In addition, the 2 × 4 ANOVA revealed a significant interaction between Group and Temperament, F = 3.994, p = 0.013, ηp2 = 0.028. A simple effects analysis indicated that the sanguine temperament was significantly higher than the scores for the other three temperaments within the dancers and these  are illustrated with designation of any differences in p < 0.05  = 54.053, p < 0.001, ηp2 = 0.275. Specifically, the frontal lobe exhibited greater power relative to the other lobes in all study participants, all ps < 0.001, and that the central region exhibited greater power than the parietal, temporal and occipital lobes, ps < 0.01.The 2 (Group: dancer vs. nondancer) × 2 (Hemisphere: left vs. right) × 5  ANOVA revealed a significant effect of Region, F = 30.920, p < 0.001, ηp2 = 0.289, such that Fz exhibited the greatest theta power and Oz exhibited the smallest, ps < 0.05.The 2 (Group: dancer vs. nondancer) × 4  ANOVA revealed a significant effect of Midline location, F = 6.489, p < 0.001, ηp2 = 0.044, such that the temporal lobes exhibited lower low-alpha power than that exhibited in the frontal, parietal and occipital lobes, ps < 0.05.The 2 × 2 × 5 ANOVA revealed a significant main effect of Region, F = 0.141, p = 0.935, group, F = 1.327, p = 0.251, or any interaction between Group and Midline location, F = 0.146, p = 0.932.The 2 × 4 ANOVA revealed no effect of Midline location, F = 8.092, p < 0.001, ηp2 = 0.054, such that the occipital lobe exhibited greater power than that observed in the frontal, central and temporal lobes, ps < 0.01, while the parietal lobe exhibited greater power than that in the temporal lobe, p = 0.019.The 2 × 2 × 5 ANOVA revealed a significant effect of Region, F = 0.863, p = 0.461, group, F = 0.001, p = 0.981, or interaction between Group and Midline location, F = 0.244, p = 0.866.The 2 × 4 ANOVA revealed no effect of Midline location, F = 4.05, p = 0.045, ηp2 = 0.007, such that dancers exhibited higher low-beta power than nondancers. Additionally, the 2 (Group) × 4 (Midline location) ANOVA also revealed an effect of Group, F = 3.876, p = 0.050, ηp2 = 0.017, such that dancers exhibited greater power than nondancers (The 2 (Group) × 2 (Hemisphere) × 5 (Region) ANOVA revealed that the effect of Group was significant, ndancers .F = 7.091, p = 0.008, ηp2 = 0.012, such that dancers exhibited greater high-beta power than nondancers. An interaction between Group and Region was also significant, F = 2.619, p = 0.034, ηp2 = 0.018. A simple effects analysis revealed that the dancers revealed greater magnitude of difference in power in the occipital lobe than nondancers did (p < 0.001). In addition, within the dancer group, the occipital lobe exhibited greater power than the frontal, central, parietal  and temporal regions (p = 0.048); and the temporal lobe revealed greater high-beta power than the central and parietal regions, values of p = 0.007 (The 2 (Group: dancer vs. nondancer) × 2 (Hemisphere: left vs. right) × 5  ANOVA revealed a main effect of Group,  = 0.007 .F = 7.028, p = 0.009, ηp2 = 0.030, such that dancers exhibited greater high-beta power than nondancers. The interaction between Group and Midline location was also significant, F = 3.089, p = 0.028, ηp2 = 0.039. The simple effects analysis revealed that dancers exhibited greater power in the occipital lobe than nondancers (p < 0.001). Finally, dancers exhibited greater high-beta power in the occipital than the central, frontal, and parietal lobes, all values of p < 0.001 (The 2 (Group: dancer vs. nondancer) × 4  ANOVA also indicated that the effect of Group was significant,  < 0.001  and 2C.r = −0.447, p = 0.015), Cz , Pz  and Oz . Such a directional relationship was also found within dancers for low-beta power at the Oz electrode site .Within dancers, the preferred tempo was negatively correlated with high-beta power at midline electrode sites Fz . Dance is a type of movement embraced by individuals to express emotion or affect; however, the musical tempo preference and temperament traits of dancers have received little attention in the literature. Therefore, the present study addressed this gap and, furthermore, subscribed to a multilevel analysis by additional consideration of psychophysiological assessment of neural activity during melodic recall. Relative to nondancers, we observed an extraverted temperament  in ballroom dancers accompanied by a preference for a faster musical tempo. This was complemented by the finding of elevated EEG beta activity, indicative of heightened cerebral cortical arousal, during their melodic recall. These results are consistent with arousal theory , suggestAlthough the dancers showed a temperamental profile supportive of extraversion they also demonstrated, paradoxically, an element of heightened introversion  compared to nondancers, which appears contradictory. Such a conundrum may be resolved by consideration of interactional personality theory , which eAs shown in The finding of a higher tempo preference in dancers vs. nondancers also supports those of previous studies of the perception of dance music , 2008. BIn support of the arguments above, Although the associations found between neural activity and personal traits has a long history , the linAlthough devoid of main or interactive effects with group membership , the regional topographical effects noted for EEG theta, low-alpha and high-alpha band power contribute to confidence in the findings based on the distribution of regional activity. For example, a significant main effect of elevated frontal theta, relative to all other regions, was noted during melodic recall. Such an effect implies the engagement of working memory during the tapping task in all the study participants while the regional distribution of elevated high-alpha power in the occipital region is consistent with the typically topography of EEG alpha band power .In summary, an association between personality and music preference was previously reported by several groups . AccordiThe present experimental paradigm employed a new tapping task, one that allowed participants to freely recall their preferred tempo and tap that tempo on a keyboard. This method differed from the category-based and self-report methods used in previous publications . Our intThe present results are consistent with arousal theory, suggesting that dancers need more stimulation and choose active and energetic music (faster tempo), providing the nervous system with higher arousal levels. To our knowledge, the present study is the first to use a modified voluntarily tapping task to examine tempo preference in a natural manner. Moreover, the study focused on a specific population within the performing arts and subscribed to a multilevel approach consisting of measures of temperament and psychophysiology, thus providing an integrated perspective of the tempo preference of dancers. The finding that temperament was associated with heightened brain activity during musical recall raises the possibility of a mechanistic relationship that offers a promising line of investigation for future studies. A fundamental question is whether the observed preference in tempo is learned as a result of practice and experience or, conversely, whether a dancer’s dispositional tendencies contribute primarily to tempo preference. In essence, is it learning or gravitation driven by personality that primarily drives the engagement to achieve skill and high levels of human performance? This is an age-old question, but the present investigation supports a role, whether primary or not, of temperament in human performance.10.7717/peerj.10658/supp-1Supplemental Information 1Each data point indicates the tempo preference and score of temperament of each participants.Click here for additional data file.10.7717/peerj.10658/supp-2Supplemental Information 2Each data point indicates the power of theta, low alpha, high alpha, low beta and high beta in each region and hemisphere for each participants.Click here for additional data file.10.7717/peerj.10658/supp-3Supplemental Information 3Each data point indicates the code in raw dataset file and their paraphrase.Click here for additional data file.


To evaluate the possible synergic effect of cisplatin and low molecular weight heparin (LMWH) on oral squamous cell carcinoma (OSCC).Cisplatin and enoxaparin sodium, alone or in combination, were administered at doses of 1, 2, 4, 8 and 10 µM and 0.1, 0.5, 1, 5, 10, 50, and 100 µg/ml, respectively, to the H357 human OSCC line. The effects on cell viability and apoptosis were evaluated after 24, 48, and 72 h and on cell migration after 18 and 24 h.p=0.009), 48 (p=0.001) and 72 h (p = 0.003); the 100 µg/ml dose of enoxaparin produced the greatest decrease in cell viability but without significant differences (p>0.05). When different concentrations of cisplatin and enoxaparin were combined, it was found that 100 µg/ml enoxaparin sodium produced the greatest synergic effect on cell viability reduction. In analyses of apoptosis and cell migration, it was found that the combination of cisplatin at 8 or 10 μM and 100 μg/ml enoxaparin produced a higher rate of apoptosis at 24, 48, and 72 h and a greater reduction in cell migration at 18 and 24 h.10 µM concentration of cisplatin produced the greatest decrease in cell viability, with significant differences at 24 (A combination of cisplatin and enoxaparin sodium shows a synergic effect that reduces cell viability and cell migration capacity and increases the apoptosis of human OSCC cells.Enoxaparin may be beneficial in chemotherapy for patients with OSCC; this finding requires further clinical and laboratory investigation. Cancer is the main cause of death in both the developed and the developing worlds. It is predicted that numbers of death resulting from cancer will grow as populations and life expectancy increase, especially in the developing world where 82% of the world’s population is located. In the least developed countries, lifestyle habits that constitute risk factors for developing cancer are spreading – smoking, alcohol consumption, a nutritionally poor diet (low consumption of fruit and vegetables), physical inactivity (obesity), and changing reproductive habits  – and the numbers of cases of cancer have increased . SquamouConventional chemotherapy for OSCC is based on cisplatin (cis-diammine-dichloro-platinum), the first of a family of drugs that currently include carboplatin, oxaliplatin, satraplatin, and picoplatin. Among medical cancer treatments, chemotherapy with cisplatin has the greatest impact and its introduction has changed the therapeutic management of a range of tumors over the last 40 years. These include cancers of the bladder, breast, lung, lymphomas, testicles, ovaries , as wellIn addition to the adverse effects of this drug  , there avia two paths; on the one hand, by impeding thrombin generation, which inhibits the tissue factor pathway through the release of an endothelial tissue factor (TF) pathway inhibitor (TFPI) (Low molecular weight heparins (LMWHs) were approved by the US Food and Drug Administration (FDA) in 1998 as an anticoagulant treatment and have been administered satisfactorily ever since . More rer (TFPI) , and on r (TFPI) . Its antr (TFPI) –40.Enoxaparin sodium is an LMWH obtained by an alkaline depolymerization method; it has an average molecular weight of 4.5 kDa, and its anticarcinogenic activity has been studied in cases of pancreatic adenocarcinoma cells, human breast carcinoma cells, human lung adenocarcinoma epithelial cells, glioma cells, melanoma cells , 43–47 aThe aim of this study was to evaluate the possible synergic effect of cisplatin and enoxaparin sodium on OSCC.2.The study used the H357 human OSCC line (European Collection of Cell Cultures), belonging to stage 1 OSCC (T1N0M0) located at the base of the tongue of a male patient. Cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal calf serum (FCS), glutamine (2 mM), 0.5 μg/ml hydrocortisone sodium succinate, 1% penicillin, and 1% streptomycin (full medium) at 37°C, in an atmosphere of 95% oxygen and 5% CO® .The medium (IMDM), 3--2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), cisplatin, and enoxaparin sodium used in the study were supplied by Sigma-AldrichCisplatin was dissolved in 0.5% DMSO and enoxaparin sodium in phosphate buffered saline (PBS), with 1 mg/ml of cisplatin or enoxaparin sodium being used as a stock solution. The working solutions were diluted with Iscove’s modified Dulbecco’s medium (IMDM). All manipulations with cisplatin and enoxaparin sodium were performed under subdued lighting. The dose range was 1, 2, 4, 8 and 10 µM of cisplatin and 0.1, 0.5, 1, 5, 10, 50, and 100 µg/ml of enoxaparin sodium.The technique described by Carmichael et al. , 51 was At different time points after the start of treatment , the medium was eliminated and the cells were incubated with MTT  (1 mg/ml) for 4 h, after which the non-metabolized MTT was discarded and 100 µl of DMSO were added to each well. Absorbance in each well was measured with an enzyme-linked immunosorbent assay (ELISA), using a Multiskan MCC/340P plate spectrophotometer at a reading wavelength of 570 nm and a reference wavelength of 690 nm. Each test was performed in triplicate.The ELISA cell death detection kit was used (following the manufacturer’s instructions) to detect apoptosis in cells treated with cisplatin and enoxaparin sodium. Brieﬂy, cells were seeded in 96-well plates at a density of 3,200 cells per well for 24 h, adding the medium containing the two highest concentrations of cisplatin used in the cell viability test (8 and 10 µM) combined with the highest concentration of enoxaparin sodium used in the cell viability test (100 µg/ml). After 24, 48, or 72 h, the cytoplasm inthe control and treatment groups was transferred to the 96-well plate, peridiumed by streptavidin, and incubated with biotinylated histone antibody and peroxidase-tagged mouse anti-human DNA for 2 h at room temperature. Absorbance at 405 nm was measured with EXL-800 type Enzyme-Linked Immunosorbent apparatus. Each test was performed in triplicate.® image software  and applying the formula: migration distance = initial distance of free-of-cells space – distance at 18 or 24 h of free-of-cells space . A descriptive study was made of each variable. The associations between different quantitative variables were studied using one-way analysis of variance (ANOVA) for more than two samples, verifying in each case whether variances were homogeneous. Statistical significance was accepted for p ≤ 0.05.Data were analyzed using the SPSS version 20.0 statistical software package (SPSSp=0.009), 48 h (p=0.001), and 72 h (p=0.003) (p= 0.215), 48 h (p=0.558), or 72 h (p=0.303) incubation , it was found that as the dose of cisplatin increased, OSCC cell viability decreased. The 10 μM cisplatin concentration produced the greatest reduction in cell viability, with statistically significant differences at 24 h , and for 1, 2, 4 and 8 µM cisplatin at 48 h incubation   were combined with different concentrations of enoxaparin sodium  it was found that combining any concentration of cisplatin with 100 µg/ml enoxaparin sodium produced the greatest synergic effect OSCC cell viability reduction, with statistically significant differences for combinations of 8 and 10 µM cisplatin at 24 h incubation (ctively) 3.Both the cell death test and the cell migration assay, investigated the two highest concentrations of cisplatin (8 and 10 µM), and enoxaparin sodium (100 µg/ml), as these doses led to the greatest reductions in cell viability.p=0.008 and p=0.009, respectively)  Figure 4p=0.003) and 24 h (p=0.004)  7.Most tumors in the oral cavity, pharynx and larynx (>90%) are squamous cell carcinomas. OSCC represents 6% of all malign neoplasias and constitutes the eighth most common cancer in terms of worldwide incidence . MortaliCisplatin is the most often used chemotherapy in OSCC treatment, often administered in combination with taxanes and/or 5-fluoruracil . But in Heparin and LMWHs have shown substantial anticarcinogenic properties in addition to their traditional anticoagulant properties , 58, 59.The present study used enoxaparin sodium, which is an LMWH whose anticarcinogenic activity has been investigated inpancreatic adenocarcinoma cells, human breast carcinoma cells, human lung adenocarcinoma epithelial cells, glioma cells, melanoma cells , 43–47, The present study assayed the effect of enoxaparin sodium on cell viability at 24, 48 and 72 h incubation, observing that, as the dose of LMWH increased, cell viability decreased, with the greatest reduction found with the 100 μg/ml dose, although no statistically significant differences were found at any of the incubation times assayed. Nevertheless, in 2011, Abu Arab et al.  observedp<0.001 and p<0.001, respectively), and for 1, 2, 4 and 8 µM cisplatin at 48 h incubation . The present results for the action of cisplatin in combination with enoxaparin sodium up OSCC cells cannot be compared with any previous investigation of the possible synergic effects of these drugs for treating OSCC. Nevertheless, in 2016, Djaafar et al. . In 2006, Balzarotti et al.  and 24 h (p=0.004) incubation. The interaction of enoxaparin sodium with heparanase at the start of the tumor metastasis process would appear to be closely related to the phenomenon of reduction in cell migration. During this step in the process, carcinogenic cells degrade the extracellular matrix and the basal membrane (including its main components—heparan sulfate proteoglycans [PGHS]) through heparanase, subsequently releasing cytokines, chemokines, and angiogenic growth factors ), so favoring angiogenesis, tumoral growth and metastasis. However, the reduction in heparanase expression (overexpressed in most human tumors) by the action of enoxaparin sodium will reduce this cell migration mechanism. In a study by Djaafar et al. (Lastly, when 8 or 10 μM cisplatin were combined with 100 μg/ml enoxaparin sodium, this produced the greatest reduction in cell migration capacity, with statistically significant differences for 8 μM cisplatin with 100 μg/ml enoxaparin sodium, at both 18 h (r et al. , treatmer et al.  using thr et al.  who studIn conclusion, the combination of cisplatin and enoxaparin sodium showed a synergic effect in reducing cell viability and migration capacity and increased the apoptosis of H357 human OSCC cells. The present results suggest enoxaparin sodium could be beneficial in chemotherapy for OSCC patients. Further laboratory and clinical assays should be conducted to confirm and develop the present findings see 2.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.All authors have worked in an equal way in research duties for developing this work. The authors declare that there is no any conflict interest regarding this submission. All authors contributed to the article and approved the submitted version.The fee will be paid with the grant of University of Murcia provided to Department of Oral Surgery. This paper is partially supported by Ministerio de Ciencia, Innovación y Universidades grant number PGC2018-097198-B-I00 and Fundación Séneca de la Región de Murcia grant number 20783/PI/18.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.


Botrytis cinerea) is a fungal plant pathogen causing postharvest decay in strawberry fruit. Here, we conducted a comparative transcriptome analysis to identify differences in gene expression between the immature-green (IG) and mature-red (MR) stages of the “Sunnyberry” (gray mold-resistant) and “Kingsberry” (gray mold susceptible) strawberry cultivars. Most of the genes involved in lignin and alkane-type wax biosynthesis were relatively upregulated in “Sunnyberry”. However, pathogenesis-related proteins encoding R- and antioxidant-related genes were comparatively upregulated in “Kingsberry”. Analysis of gene expression and physiological traits in the presence and absence of B. cinerea inoculation revealed that the defense response patterns significantly differed between IG and MR rather than the cultivars. “Kingsberry” showed higher antioxidant induction at IG and upregulated hemicellulose-strengthening and R genes at MR. Hence, “Sunnyberry” and “Kingsberry” differed mainly in terms of the expression levels of the genes forming cuticle, wax, and lignin and controlling the defense responses. These discrepancies might explain the relative difference between these strawberry cultivars in terms of their postharvest responses to B. cinerea.Gray mold ( Fragaria × ananassa Duch.) is an economically important horticultural crop. The export value of strawberries in Korea increases annually. Strawberry production and exportation have risen more than threefold over the past two decades [Strawberry  as well as phytotoxic compounds to facilitate host penetration and infection [ tissues . When B.n hyphae . B. cinenfection .B. cinerea inoculation in mature-red (MR) strawberry fruit. Complex crosstalk among jasmonic acid, salicylic acid, ethylene, and abscisic acid participates in defense responses by regulating defense-, ripening-, and senescence-related genes [Plants have evolved defense mechanisms against postharvest fungal pathogens . Cuticleed genes ,15. Reaced genes .B. cinerea is quiescent in green fruit [Next-generation sequencing identifies factors that contribute to fungal pathogen resistance in horticultural crops. Several researchers have attempted to identify the differences between resistant and susceptible cultivars in terms of defense response ,18,19. Cen fruit ,23,24.B. cinerea. Studies on various B. cinerea-resistant strawberry cultivars have been conducted. An earlier work assessed postharvest storability in five different strawberry cultivars developed in Korea [Resistance to postharvest fungal decay in strawberries depends largely on the genetic background of the cultivar. Dong et al.  reportedin Korea . The “SuB. cinerea. Here, we compared the transcriptomes of the aforementioned “Sunnyberry” and “Kingsberry” strawberry cultivars. We characterized their differentially expressed genes (DEGs) involved in pre-formed and induced defense responses. We determined the relative expression levels of several genes by quantitative polymerase chain reaction (qPCR) analysis. We evaluated the physiological traits of these fruit varieties in the presence and absence of B. cinerea inoculation. This study will help clarify the various defense responses in two different strawberry cultivars and identify the genetic factors that probably contribute to B. cinerea resistance in this crop. Our findings will also provide a theoretical basis for selecting and breeding strawberry cultivars with enhanced postharvest fruit storability.To the best of our knowledge, no prior research has compared the transcriptomes of strawberry cultivars with different responses to B. cinerea at the MR stage  and MR stages were compared. Fifty-five million reads per sample were obtained through RNA sequencing (RNA-Seq) . In “SunGenes related to cell wall metabolism were differentially expressed between the “Sunnyberry” and “Kingsberry” cultivars . At IG, O-methyltransferase, cinnamoyl-CoA reductase 1 (CCR1), and cinnamyl alcohol dehydrogenase (CAD) genes were upregulated in “Sunnyberry” compared with those in “Kingsberry”. In contrast, at IG and MR, caffeoyl-CoA O-methyltransferase genes were upregulated in “Kingsberry” relative to “Sunnyberry”. Certain CAD genes were annotated as putative mannitol dehydrogenases. Nevertheless, these genes may be functionally redundant and could be considered CAD [POD and LAC were upregulated in “Sunnyberry” compared with those in “Kingsberry”.Here, we identified DEGs involved in lignin biosynthesis . At bothered CAD . Class IHCD was comparatively upregulated in “Kingsberry”. However, there was no trend in the expression of any other DEGs involved in fatty acid elongation. Protein ECERIFERUM 1-like (CER1) genes mediate decarbonylation of very long-chain fatty acids (VLCFAs) and were comparatively upregulated in “Sunnyberry” at IG and MR. Wax ester synthase/diacylglycerol O-acyltransferase (WSD) genes mediate acyl reduction of VLCFAs and were relatively upregulated in “Kingsberry” at both stages.Cuticular wax is biosynthesized by fatty acid elongation, decarbonylation, and acyl reduction . Here, wArachis diogoi Hoehne), TLP was upregulated after Phaeoisariopsis personata (late leaf spot fungus) inoculation. In vitro, recombinant TLPs presented with activity against several fungal pathogens, including B. cinerea [Certain pathogenesis-related (PR) genes were identified as DEGs . At MR,  cinerea .At both IG and MR, most DEGs encoding RPM 1-like protein and RPP13-like protein 1 were relatively upregulated in “Kingsberry”. Several bioinformatically predicted resistance gene analogs  were alsAt IG and MR, the antioxidant peroxiredoxin (PRX) and superoxide dismutase were comparatively upregulated in “Kingsberry” . The thiB. cinerea inoculation. Their relative expression levels were measured by qPCR (Eight DEGs were selected at the IG and MR stages of “Sunnyberry” and “Kingsberry” in the presence or absence of  by qPCR . In tota by qPCR C.CAD was upregulated after B. cinerea inoculation only at IG while cellulose synthase-like protein D3 (CSLD3) was upregulated after B. cinerea inoculation only at MR. At MR, CCR1 was relatively upregulated in “Sunnyberry”. However, B. cinerea inoculation dramatically lowered CCR1 mRNA in both cultivars at MR.In both cultivars, B. cinerea inoculation, CER1 was upregulated in “Sunnyberry” compared with that in “Kingsberry” at IG and MR. The opposite was true after B. cinerea inoculation. LACS4 was upregulated after B. cinerea inoculation only at MR. In the presence and absence of B. cinerea inoculation, LACS4 had higher expression levels in “Kingsberry” than in “Sunnyberry” at MR.In the absence of βGLU presented with very wide SD. Hence, it did not significantly differ between cultivars. In the presence and absence of B. cinerea inoculation, TLP1b was relatively upregulated in “Sunnyberry” but was significantly suppressed at MR by inoculation. RPM1 was comparatively upregulated in “Kingsberry” at IG and MR but its relative mRNA level was substantially higher at MR. RPM1 was upregulated in MR fruits after B. cinerea inoculation. “Kingsberry” showed higher relative RPM1 mRNA levels after B. cinerea inoculation.The relative mRNA level of 2O2) content, total phenolic content, and total antioxidant activity were determined in the presence and absence of B. cinerea inoculation  and one that is susceptible to this fungal pathogen (“Kingsberry”). We also measured the relative expression levels of eight selected DEGs and compared the physiological traits of these strawberry varieties in the presence and absence of B. cinerea inoculation.In the present study, we evaluated the transcriptomic differences between a strawberry cultivar showing resistance to B. cinerea penetration and invasion [B. cinerea. PG and PL were relatively upregulated in “Sunnyberry” while PGL3, PAE, and PME were comparatively upregulated in “Kingsberry”  was obtained from the Center for Fungal Genetic Resources at Seoul National University, Seoul, South Korea, and cultured on potato dextrose agar at 25 °C. A conidial suspension was prepared using a 3-week culture. The conidia were collected with 2 mL sterile distilled water and filtered through four layers of gauze. The concentration of the conidial suspension was evaluated using a hemocytometer  with a DE/Axio Imager A1 microscope  and adjusted to 1 × 105 mL−1.v/v) sodium hypochlorite for 3 min, rinsed in sterile distilled water for 2 min, and dried for 2 h on a clean bench. Each fruit was inoculated by dropping 10 μL conidial suspension onto its receptacle. A total of fifteen fruits for each stage were used for the inoculation, and the inoculated fruits were then stored at 25 °C and 100% relative humidity n a plastic box sealed with Parafilm® .IG and MR strawberry fruits were disinfected by immersion in 2%  was calculated by dividing the number of decayed fruits by the total number of fruits and multiplying the quotient by 100. The disease incidence rate was calculated twice in 2019 and 2020. B. cinerea and stored at 10 °C for 10 d, and their disease indices were rated as follows: 0, no symptoms; 1, symptoms on 0–10% of the fruit surface; 2, symptoms on 10–25% of the fruit surface; 3, symptoms on 25–50% of the fruit surface; 4, symptoms on >50% of the fruit surface  CTAB, 2.5 M NaCl, 300 mM Tris HCl (pH 8.0), 25 mM EDTA, and 2% (w/v) PVP) and the suspension was shaken vigorously. The sample was incubated in a drying oven at 65 °C and shaken vigorously every 5 min. The suspension was extracted twice with equal volumes of 24:1 (v/v) chloroform:isoamyl alcohol and centrifuged at 3500× g and 4 °C for 15 min. The top layer was transferred to a new tube and centrifuged at 25,000× g and 4 °C for 20 min. The supernatant was mixed with 0.1 vol of 3 M NaOAc and 0.6 vol isopropanol and stored at −20 °C for 30 min. The sample tube was centrifuged at 3500× g and 4 °C for 30 min to collect the nucleic acid pellet. The pellet was dissolved in 1 mL Tris-EDTA and mixed with 0.3 vol of 10 M LiCl.Fruit samples 0.1 g in weight were flash-frozen in liquid nitrogen and pulverized. Total RNA was extracted from the powders with a Ribospin Seed/Fruit total RNA isolation kit  according to the manufacturer’s instructions. Total RNA was extracted from MR fruit inoculated with d et al. . Each 0.g and 4 °C for 30 min. The pellet was washed with 70% (v/v) ethanol and centrifuged at 3500× g and 4 °C for 5 min. The supernatant was discarded, and the pellet was centrifuged at 3500× g and 4 °C for 1 min to evaporate any remaining ethanol. The pellet was dissolved in 50 μL sterile distilled water. All solvents except the extraction buffer were ice-cold. RNA purity was assessed by gel electrophoresis. The A260:A230 and A260:A280 ratios were measured in a microplate spectrophotometer .The RNA was precipitated overnight by incubation at 4 °C and collected by centrifugation at 20,000× The total extracted RNA was used for RNA-Seq and qPCR analyses. For qPCR, cDNA was synthesized from 500 ng total RNA with a ReverTra Ace qPCR RT kit  according to the manufacturer’s instructions.Fragaria × ananassa octoploid “Camarosa” annotated genome [p < 0.01 and |fold-change| > 2. GO functional analysis was conducted using Blast2GO v. 5.2 and the NCBI  database [Total RNA from the IG and MR stages of “Sunnyberry” and “Kingsberry” strawberry fruit was used. RNA-Seq was performed at the National Instrumentation Centre for Environmental Management of Seoul National University  on a HiSeq 2500 platform  using 151 bp paired-end reads. Low-quality (<Q20), adaptor, and barcode sequences were trimmed out with Trim Galore v. 0.4.4 using the parameter “—gzip–paired”. Cleaned reads were mapped to the d genome . To idendatabase .−1 and combined with 5 μL of 2 × Real-Time PCR Master Mix containing SYBR Green 1 . PCR primers (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). For each reaction, relative gene expression was normalized to the expression of the reference gene elongation factor 1-alpha (maker-Fvb3-3-snap-gene-215.43) by the 2−ΔΔCt method [qPCR analysis was conducted as described by Min et al. . The cDN primers  were dest method .2O2 content was determined according to the method of Junglee et al. [w/v), and incubated at 4 °C for 10 min. All samples were centrifuged at 12,000× g and 4 °C for 20 min. Each 0.5 mL supernatant was mixed with 0.5 mL of 1 M KI and 0.25 mL of 10 mM potassium phosphate buffer (pH 7.0). The mixtures were incubated in the dark at 22 °C for 20 min. Absorbance was measured at 390 nm using a microplate spectrophotometer . A standard curve was plotted using various H2O2 concentrations.The He et al. , with slv/v) methanol, vortexed for 15 s, sonicated for 20 min, and centrifuged at 3000× g and 22 °C for 20 min. Each supernatant was transferred to a new 15 mL tube, diluted with an equal volume of 80% (v/v) methanol, and used in the subsequent assays.Each 0.2 g sample was flash-frozen in liquid nitrogen, pulverized, combined with 10 mL of 80% (w/v) Na2CO3 and 200 μL distilled water were added, and the mixture was incubated in the dark at 22 °C for 30 min. Absorbance was measured at 750 nm using a microplate spectrophotometer . A standard curve was plotted using various gallic acid dilutions.Total phenolic content was determined with Folin–Ciocalteu reagent . Fifty m•+ scavenging activity [•+ solution was diluted with distilled water to OD734 = 0.7. Then, 1.2 mL diluted ABTS•+ solution was mixed with 10 μL sample. The mixture was vortexed briefly and incubated in the dark at 22 °C for 15 min. Absorbance was measured at 734 nm using a microplate spectrophotometer . A standard curve was constructed using various Trolox dilutions.Total antioxidant activity was determined by evaluating the ABTSactivity . The ABT280 g FW-1.Lignin was quantified according to the method of Ring et al. . Analyse2O2, total phenolic, and lignin content and total antioxidant activity were determined using four biological replicates (n = 4). Significant differences between treatment means were determined with a two-sample t-test in SPSS v. 25.0 .RNA-Seq and qPCR analyses were conducted using three biological replicates (n = 3). HB. cinerea. The DEGs between “Sunnyberry” and “Kingsberry” at IG and MR disclosed that “Sunnyberry” exhibited relatively higher expression levels of genes related to lignin and cuticle biosynthesis. In contrast, “Kingsberry” displayed higher expression levels of genes regulating PR, R, and antioxidant system-related proteins. Examination of induced defense responses revealed that the physiological traits and the expression levels of genes controlling cell wall metabolism, cuticle biosynthesis, and defense response varied with cultivar and developmental stage. Our study demonstrated that “Sunnyberry” and “Kingsberry” differed in terms of their pre-formed physical barriers and induced defense responses. However, their defense strategies were effective at MR and contributed to B. cinerea resistance.In the present study, we investigated transcriptomic differences between two strawberry cultivars with varying degrees of resistance to


Trichogramma achaeae Nagaraja and Nagarkatti (Hymenoptera: Trichogrammatidae) is currently used as biological control agent for several lepidopteran pests. Knowledge of thermal requirements is essential to optimize its rearing procedures and inundative releases. The biological characteristics and two-sex life table parameters of T. achaeae were determined at five constant temperatures  using Ephestia kuehniella Zeller  eggs. T. achaeae was able to develop and survive from 15 °C to 30 °C, but not at 35 °C. Temperature significantly affected the preadult development time and adult longevity, decreasing when temperature increased from 15 °C to 30 °C. Temperature significantly altered the sex ratio, being female biased between 15 °C and 25 °C. Age-stage, two-sex life table analysis indicated that net reproductive rate (R0) was highest at 20 °C. Both the intrinsic rate of increase (r) and finite rate of increase (λ) increased with increasing temperature, while the mean generation time (T) decreased significantly. In addition, functional response of T. achaeae was studied, being significantly affected by temperature and host egg density, displaying a Holling type-I at 15 °C and a Holling type-II at 25 °C. The relevance of these results is discussed for the use of T. achaeae as biological control agent. Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) has expanded rapidly since its accidental introduction in eastern Spain in 2006 [Chrysodeixis chalcites (Esper) (Lepidoptera: Noctuidae) is an extremely polyphagous species that has increased its movement and spread within central and northern Europe, causing severe damage to horticultural and ornamental crops both in open fields and in greenhouses [Global trade and climate change have led to an exponential increase in the introduction and establishment of numerous invasive insect pests to new geographic areas, producing substantial ecological and economic impacts . Several in 2006 . In a fe in 2006 ,4,5. Besenhouses , which ienhouses .The appearance of lepidopteran pests in newly invaded areas has been predominantly managed by extensive application of broad-spectrum and persistent insecticides ,9, whichTrichogramma (Hymenoptera: Trichogrammatidae) has shown efficient results in controlling several lepidopteran pests in many agricultural crops and forests worldwide [Trichogramma achaeae Nagaraja and Nagarkatti have been reported as a potential biological agent for controlling T. absoluta and C. chalcites eggs in the Palearctic region [T. achaeae is currently commercialized and used in some European and North African countries against different Lepidoptera species in more than 15 horticultural and ornamental crops [T. achaeae has been shown to be a highly efficient biological control agent of T. absoluta in some Mediterranean Basin countries when is used in combination with inoculative releases of the omnivorous predator Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) in commercial tomato greenhouses [T. achaeae have showed promising control results of C. chalcites in banana crops of the Canary Islands when they are combined with pest monitoring and additional application of biological insecticides as the entomopathogenic bacteria Bacillus thuringiensis var. kurstaki [T. achaeae has also been successful released against Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) and other bollworms species in cotton crops in India [Among these strategies, inundative biological control with egg parasitoids of the genus orldwide ,14,15. Torldwide , reducinorldwide . Trichogc region . It is ac region , but it c region . This pac region ,18 and hc region  and the c region . In addial crops ,22. Indeenhouses ,23,24,25kurstaki . Additioin India .Trichogramma wasps in biological control programs is determined by some important factors such as the potential development of the parasitoid species or strain in the target host, beside some abiotic and physical factors [Trichogramma [Trichogramma species/strain is also essential for planning its mass rearing procedures in laboratory [Trichogramma depends on species/strains studied [The successful selection and introduction of  factors ,28,29. Ahogramma , becausehogramma ,32. Undeboratory ,33 and tboratory . However studied ,35 and t studied ,36.Trichogramma species as biological control agent is the number of parasitized eggs in response to the parasitoid and host densities, i.e., its functional response [Trichogramma species usually present a type I or type II functional response [Another essential factor determining the selection and efficiency of a response , definedresponse  as the rresponse . Followiresponse , the genresponse ,42. In tresponse ,44,45,46response ,48 or thresponse .T. achaeae is poorly studied [T. absoluta [T. achaeae, besides establishing its functional response in relation to temperature in Ephestia kuehniella Zeller  eggs as an alternative host in order to optimize the mass production systems and inundative releases in the field of this parasitoid species for the biological control of C. chalcites and T. absoluta, among other lepidopteran pests.Despite being a high economic importance species, the biology of  studied ,51,52,53 studied ,55 and iabsoluta . TherefoT. achaeae was established from parasitized eggs of C. chalcites originally collected on banana crops in the locality of El Remo, La Palma, Canary Islands . Emerging wasps were successfully identified by examination of morphological characters and molecular characterization by sequencing of the ITS2 gene [T. achaeae was reared for at least 15 generations before starting the trials, using fresh ultraviolet-sterilized eggs of E. kuehniella as factious host in a climatic chamber , according to the methodology proposed by Cabello et al. [E. kuehniella massively reared following the methodology defined by Cerutti et al. [A laboratory colony of TS2 gene ,21. T. ao et al. . Factioui et al. . A singli et al. , before T. achaeae was studied at 15, 20, 25, 30, and 35 ± 1 °C, with a 60–80% RH and a 16:8 h L:D photoperiod in climatic chambers. From each temperature, newly emerged pairs  of T. achaeae were randomly chosen from the laboratory colony and exposed to fresh ultraviolet-sterilized eggs of E. kuehniella in small glass vials (5 × 1 cm). After 24 h, all parasitoids were removed, and 100 parasitized eggs (<24 h old) were individually isolated in glass vials with help of a wet fine brush (no. 0) and transferred to temperature-controlled chambers. Each isolated egg represented a replicate. Parasitized eggs were daily examined until the parasitoids completed their development and the sex of emerging wasps was recorded. Because larval development of T. achaeae occurs within the host egg, the immature stages  were regarded as preadult stage (egg–pupa).The egg to adult developmental time of T. achaeae adults were also evaluated at 15, 20, 25, 30, and 35 ± 1 °C, with a 60–80% RH and 16:8 h L:D photoperiod. For each temperature, 20 male/female pairs less than one day old (<24 h) obtained in the development studies were separately isolated in small glass vials covered with cotton and a water–honey (1:1) drop for feed. Cardboards (2 × 2.5 cm) with approximately 100 fresh irradiated E. kuehniella eggs were supplied daily until the natural death of the adults. Parasitized host eggs were removed daily and incubated at the above-mentioned conditions until the offspring emergence. Adult longevity, fecundity , as well as the emergence rate , and the sex ratio of the progeny  were recorded.The longevity and fecundity of T. achaeae was determined at 15, 25 and 35 ± 1 °C, with a 60–80% RH and 16:8 h L:D photoperiod following the methodology described by Cabello et al. [T. achaeae without previous parasitation experience, and E. kuehniella eggs (less than 24 h old) were used. For each test , the experimental design was totally randomized with only one factor—host density at five levels , carrying out 10 repetitions for each level of treatment. One mated female of T. achaeae was used per replicate. Host eggs were attached using a wet fine brush to cardboards (3 × 1 cm) in rows and columns separated 2 mm. Adult females were individually isolated in small glass vials and offered an egg cardboard for 24 h. Subsequently, the parasitoids were eliminated, and the egg cardboards were evolved up to offspring emergence. The registered data were parasitized eggs, emerged host larvae, and collapsed eggs.The functional response of o et al. . In all x + 1), while emergence rate and sex ratio data were transformed to arcsine (√ (x⁄100)). All transformed data were analyzed by ANOVA test applying the GLM procedure and the average values were compared by Tukey’s test (p = 0.05) by means of the statistical software IBM® SPSS® Statistics Version 22 [DT is the product of development time in days (D) and temperature (T) in degrees Celsius (°C). The intercept, k, is the thermal constant in degree days (°d), and the slope, t, the lower development threshold in °C. The parameters t and k were determined by linear regression.Development time, fecundity, and adult longevity data under different temperature regimes were transformed to log10. Life table parameters were calculated including age-stage specific survival rate (xjs) (where x is age in days and j is stage), age-stage specific fecundity (xjf), age-specific survival rates (xl), age-specific fecundity (xm), life expectancy (xje), reproductive value (xjv) and the intrinsic rate of increase (r), finite rate of increase (λ), gross reproductive rate (GRR), net reproductive rate (R0) and mean generation time (T), according to earlier theories [The raw data on e theory ,62, by mheory [® , and thetheories ,64. Boottheories  was utiltheories . A paireT. achaeae after adjustments data were submitted to two classes of statistical analysis. First, the density factor significance was determined by GLM analysis and the average values were compared by Tukey’s test (p = 0.05) by means of the statistical software IBM® SPSS® Statistics Version 22 [T. achaeae, according to the following expressions [aN is the number of parasitized hosts; tN is the density of the host or prey; a’ is the instantaneous search rate ; T is the total available search time (days); tP is the number of parasitoids; hT is the host-manipulation time; and α is parasitoid mortality potential.For the experiment concerning to the functional response of rsion 22 . Second,ressions ,67,68:(2r2) [The cited adjustments were performed by means of the software Tablecurve 2D, version 5.0 . The corr2) . HoweverT. achaeae was able to develop and emerge between 15 °C and 30 °C on E. kuehniella eggs . The developmental time was higher at lower temperatures, which ranged from 31.08 d at 15 °C to 7.55 days at 30 °C. In cultures maintained at 35 °C only emerged some adults of T. achaeae, which showed poor development of the wings and longevity less than 24 h, and none of the E. kuehniella offered eggs were parasitized, indicating that 35 °C exceeded the upper threshold. The egg-adult developmental time was not significantly different between males and females at the tested temperatures.lla eggs . StatistT. achaeae fit the linear model suggested by Ikemoto and Takai [t) was estimated in 10.51 ± 0.51 °C, while the thermal constant (k) was 138.27 ± 9.27°d .Developmental rate of nd Takai . The lowT. achaeae for each temperature tested are given in the F = 17.82, df = 3, p < 0.001) and males . In both cases, the adult longevity increased with the decrease of temperature, the maximum of 15 °C with 12.75 ± 1.22 d and 9.12 ± 1.04 days for females and males, respectively. There were significant differences in the longevity of T. achaeae females as compared to their conspecific males at the same temperature . Adult female longevity was longer than male longevity at every tested temperature . The mean number of parasitized eggs were highest at 20 °C  and lowest at 15 °C  with intermediate values in the remaining temperatures. The adult preoviposition period (APOP) and total preoviposition period (TPOP) of T. achaeae were significantly different for the tested temperatures . No APOP was observed for the female parasitoids emerged at different temperatures tested, except at 15 °C (2.15 ± 0.49 d). The females developed at 30 °C showed the shortest TPOP (7.66 ± 0.10 d), but the highest was detected at 15 °C (33.15 ± 0.49 d). Temperature also significantly influenced the mean oviposition period of T. achaeae , with the maximum at 15 °C (10.95 ± 1.13 d) and minimum at 30 °C (5.70 ± 0.37 d).The number of parasitized eggs per female (fecundity) was influenced by temperature and revealed significant differences between the tested temperatures . The highest adult emergence rate occurred at 30 °C (95.43 ± 0.64%), while the lowest was still at 15 °C (77.52 ± 2.21%). A female-biased sex ratio was recorded for T. achaeae under the temperature regimes studied. There was a significant difference in the sex ratios  at tested temperatures. The values ranged from 48.41 ± 0.91% to 68.10 ± 1.90% females at 30 °C and 15 °C, respectively.The percentage of adult emergence (fertility) was also significantly affected by temperature  was observed at 20 °C  and the lowest at 15 °C . The intrinsic rate of increase (r) of the parasitoid also increased as temperature increased, recording the lowest value at 15 °C (0.072 ± 0.005 d−1) and the highest at 30 °C (0.319 ± 0.017 d−1). The highest value for the finite rate of increase (λ) occurred at 30 °C (1.38 ± 0.02 d−1) because of the shorter duration of one generation. Finally, the mean generation time (T) decreased with increased temperature and ranged from 10.36 ± 0.10 d at 30 °C to 36.71 ± 0.39 d at 15 °C.The greatest value of the net reproductive rate (xjs) indicates the probability that an individual (as a newly laid egg) of T. achaeae will survive to age x and develop to stage j, depending on temperature is shown in T. achaeae was highest at 15 °C and lowest at 30 °C.The age-stage-specific survival rate (xl), age-stage specific fecundity (xf), and age-specific fecundity (xm) of T. achaeae reared on E. kuehniella eggs at different temperatures are illustrated in T. achaeae can successfully survive and reproduce on E. kuehniella eggs between 15 °C and 30 °C. The curves of age-specific survival rate (xl) show the probability that a newly laid egg will survive to age x. The survival rate of T. achaeae individuals at various temperatures was different. While the longest survival rate (53 d) occurred at 15 °C, its shortest value (17 d) was observed at 30 °C. The age-stage-specific fecundity (xjf) is the daily mean number of offspring produced by individual T. achaeae of age x and stage j per day. According to our results, the highest value of age-stage specific fecundity (xjf) of T. achaeae at 15, 20, 25 and 30 °C was 4.05, 22.40, 15.10 and 15.95 eggs per female and day, respectively, happening at the age of 32, 15, 9 and 8 d, respectively. The age-specific fecundity (xm) is the mean number of eggs produced per individual at age x. The age-specific maternity (xmxl) values changed depending on xl and xm, and the highest peak values of xmxl occurred at the age of 32, 15, 9 and 8 d at 15, 20, 25 and 30 °C, respectively. In accordance with the curves, the greatest fecundity happened in the two first days after female emergence.The age-specific survivorship (xje) of T. achaeae, which estimates the time that an individual of age x and stage j is expected to live. According to our results, the life expectancy of newly T. achaeae females was 12.75, 9.55, 7.55 and 6.25 d at 15, 20, 25 and 30 °C, respectively.xjv) of T. achaeae are given in x and stage j to the future population. Therefore, the reproductive value for the egg-pupa stage symbolizes the contributions of egg and pupal stages to the future population. The xjv for the egg-pupa stage of T. achaeae differed at temperatures tested, and adult female xjv peak was significantly highest at 20 °C but lowest at 15 °C, and it occurred earliest at 30 °C but latest at 15 °C. The highest adult female xvj peaks significantly raised drastically to 20.05 at 31 d at 15 °C to 45.38 at 15 d at 20 °C to 34.14 at nine d at 25 °C and to 35.23 at eight days at 30 °C.The curves of reproductive value . The greatest temperature (25 °C) can be observed to lead to higher quantities of parasitized eggs (average 16.78 eggs) than the lowest temperature .a’) was maximum at 25 °C (11.834 h−1) and minimum at 15 °C (0.8097 h−1). Host-manipulation time was 0.0414 h at 25 °C and very low at 15 °C. At 35 °C, females of T. achaeae were able to survive for 24 h; however, the percentage of parasitism found was very low, so that these values were not allowed to adjust the functional response. Also, the further development of the offspring of females did not occur to any of the densities studied. In this case, the effect was caused by the high mortality of E. kuehniella eggs at 35 °C, which collapsed and dried even when parasitized.A type I functional response corresponded to the values of the number parasitized eggs (according to density) at 15 °C and a type II functional response at 25 °C , whose aTrichogramma. In the present study, we evaluated the influence of five constant temperatures  on the biological characteristics and two-sex life table parameters of T. achaeae when it is reared in E. kuehniella eggs under laboratory conditions. Results in the current study clearly showed that temperature significantly affected the preadult development time, decreasing when temperature was increased from 15 °C to 30 °C. According to Bueno et al. [T. achaeae females and males at all tested temperatures. Similar results have been published by other authors in biological studies of different Trichogramma species [T. achaeae at 25 °C was completed in 8.83 d in E. kuehniella eggs, lower than the 10.4 d and 10.8 d recorded by Ghosh et al. [Corcyra cephalonica Stainton  and T. absoluta eggs, respectively. These differences might be associated with the effect of the size and nutritional quality of the host egg on the preimaginal development and adult fitness of Trichogramma spp., as suggested by Kishani Farahani et al. [T. achaeae developed successfully from egg to adult in the temperature range of 15 °C to 30 °C. On the other hand, the parasitized host eggs turned black at 35 °C, but many of them collapsed, dried, and no viable progeny emerged, showing a negative effect of high temperature, thus exceeding the upper threshold. The high temperature preventing the adult emergence observed in our study agrees with previous works [T. achaeae was successfully able to develop and emerge at 36 °C in C. cephalonica eggs.According to Samara et al. , temperao et al. , this sh species ,72,73. Ih et al.  and Manoh et al.  in Corcyi et al. . Our stuus works ,58. Howeus works  found thTrichogramma spp. and the best time for the release of the parasitoids in the field [T. achaeae were calculated according to linear model suggested by Ikemoto and Takai [t) and the effective accumulated temperature (k) values for egg to adult were 10.51 °C and 138.27 °d, respectively. These values were close to those previously reported for T. achaeae [Trichogramma species [T. achaeae can be successfully placed at 10 °C for short term storage up to 30 days, resulting in an adult emergence percentage of 60% at this temperature [T. achaeae to complete its development at 15 °C (31.08 d) shows that the development rate in preadult stages within E. kuehniella eggs is prolonged at lower temperatures. In this sense, earlier studies suggest that immature stages inside the host are the dominant overwintering stage of Trichogramma [T. achaeae does not enter diapause, its development being slower through the winter months [T. achaeae can cause difficulty in storage and handling, which, together with its biparental reproduction, makes the use of this parasitoid more difficult and expensive as biological control agent [Knowledge of thermal requirements allows us to expect the number of generations of he field . In the nd Takai . Our res species , suggestperature ,55. The hogramma , that cor months . On the ol agent ,56.T. achaeae. As observed in other Trichogramma species [T. achaeae females and males decreased as temperature increased, recording the highest longevity at 15 °C, for both females and males. According to Foerster et al. [T. achaeae can reproduce through the studied temperature range of 15–30 °C on E. kuehniella eggs. The highest number of parasitized eggs or fecundity was found at 20 °C  and then decreased as temperature increased. These results are according with those found for other species as T. cordubensis Vargas and Cabello [T. aurosum Sugonjaev and Sorokina [T. achaeae according to the rearing host. We found that females of T. achaeae parasitized 55.75 eggs of E. kuehniella at 25 °C, in line with the 58.4 eggs of C. cephalonica reported by Ghosh et al. [E. kuehniella, and the 22.13 eggs of T. absoluta found by Melo [Trichogramma spp. may change in accordance with the temperature [T. achaeae females, and no preoviposition period was detected at all tested temperatures, except at 15 °C. After the first day, the oviposition peak consistently declined until female death, being more notable at those temperatures in which fertility was highest. This reduction in the number of parasitized eggs over the female longevity increased is typical in pro-ovigenic species as the parasitoids of the genus Trichogramma [The current study showed that temperature significantly affected adult longevity, fecundity, parasite emergence, and sex ratio of  species ,30,75, t Cabello  and T. aSorokina , where tSorokina . On the h et al. , but hig by Melo  and Mano by Melo , respectperature  and hostperature  and foodperature . The ferhogramma .T. achaeae was relatively stable at all tested temperatures. The emergence rate values, except for those observed at 15 °C, were always above 80%, being the highest viability measured at 30 °C (95.43%). These values are greater than the previous results found for T. achaeae in C. cephalonica eggs [E. kuehniella, however it was common to find up 2–3 emerging adults from C. chalcites eggs collected in banana crops [C. chalcites eggs compared to E. kuehniella eggs and its nutritional quality for the parasitoid development [E. kuehniella eggs can be considered an appropriate alternative host to support the development of T. achaeae. Temperature is also a strongly important factor in affecting the sex ratio in Trichogramma spp. [Trichogramma [T. achaeae, being female biased between 15 °C and 25 °C, whereas it was balanced at 30 °C. Percentage of females fluctuated from 48.41 to 68.10, which is coherent with Melo [Emergence rate or viability of ica eggs , indicatica eggs . The redica eggs . A singlna crops ,21. Theselopment . Despitemma spp. ,73. Howehogramma . In our ith Melo , who notT. achaeae. Two-sex life tables are a powerful instrument to describe the probable development of a species [T. achaeae was able to quickly increase its populations in the temperature range of 15 °C to 30 °C. Several authors have adopted fertility life tables for estimating potential of Trichogramma species or strains submitted to different factors such as temperature and host adaptability [T. achaeae in response to the distinct temperature tested. The net reproductive rate (R0) varied according to the temperature variation, from 14.08 to 32.00 offspring per individual. The highest increase in net reproductive rate was observed at 20 °C. The net reproductive rate was adversely influenced by temperatures below or above 20 °C, and therefore, the population growth capacity was particularly reduced. According to Cabello and Vargas [r) and finite rate of increase (λ) were proportionally related to temperature, increasing considerably until the highest temperature was reached, while the mean generation time (T) was lowest at 30 °C. Therefore, our results indicated that E. kuehniella is an appropriate host for the mass rearing of T. achaeae, and they are in line with those reported by Varma and Maninder [C. cephalonica and T. absoluta eggs, respectively.In accordance with our knowledge, no studies have been carried out before to determine the temperature influence on population parameters of  species . Resultstability ,78. The d Vargas , the dimManinder  and ManoManinder  on C. ceT. achaeae on E. kuehniella eggs contributed essential information for the development of biological control programs, because the results demonstrated that this parasitoid species is able to survival and develop within the tested temperature range (from 15 °C to 30 °C). In this sense, Cabello et al. [T. achaeae is a species well adapted to extreme temperature conditions in commercial tomato greenhouses in southern Spain. Our results would explain the presence of T. achaeae throughout the year in banana crops in the Canary Islands, both in mesh-built greenhouses and in open fields [C. chalcites eggs are available for the parasitoid survival during all banana production cycle [T. achaeae in this study to both average minimum and maximum temperatures occurring in the Canary Islands (16–27 °C), make this species a suitable candidate as biocontrol agent of C. chalcites in banana groves [T. achaeae [Therefore, the determination of the biological characteristics, thermal requirements and population parameters of o et al.  found thn fields , where aon cycle ,79,80. Ta groves . However achaeae ,76.T. achaeae at different temperatures. The current study revealed that temperature had a substantial effect on the functional response of the egg parasitoid. Several studies have observed that the functional response of Trichogramma could change from one type to another as temperature [T. achaeae showed a type I functional response at 15 °C. In this sense, Jeschke et al. [hT) and 2) a state of maximum activity at saturation. However, in laboratory conditions at 15 °C, both requisites seem to be met for T. achaeae, who seems to have a very low hT, and only ceases to lay eggs when the female reaches its eggs in the ovarioles. These findings agree with those seen for other Trichogramma and Trichogrammatoidea species, in which most cases present type I functional responses [T. achaeae is type II, which is influenced by the time of handling. Our results are in line with a type II functional response exhibited by T. achaeae to T. absoluta eggs [T. ostrinae, the functional response is type II but changes to type III when the temperature increases from 20 °C to 27 °C [Trichogramma is the host species; thus T. pretiosum Riley parasitizing T. absoluta eggs presents a type I functional response [H. armigera eggs, the response is type II [T. chilonis Ishii that presents type II response in eggs from its natural host Chilo sacchariphagus (Bojer) changes to type III when offered eggs from its unnatural host Galleria mellonella L. [T. achaeae females must parasitize previously parasitized eggs, which is an extremely common phenomenon under natural conditions.No experiments have been performed before to determine the relationship between host densities and functional response for perature ,81 and hperature . Besidesperature . Accordie et al.  indicateesponses ,44,45,46uta eggs . Howeverto 27 °C . Besidesnella L. . On the T. achaeae, an important parasitoid of T. absoluta and C. chalcites in the Paleartic region. The age-stage, two-sex life-table analysis obtained from this research has provided fundamental information on the effects of temperature on the developmental time, survivorship, reproduction, and longevity of T. achaeae. Results showed that temperature ranging from 15 °C to 30 °C was favorable for its growth, development, and survival, as well as the adequacy of E. kuehniella eggs as alternative host in T. achaeae mass rearing systems. In addition, the results showed a clear relationship between temperature and the functional response of T. achaeae. Parasitoid female had a greater efficiency at 25 °C, showing a Holling type II functional response. The rate of parasitism increased when the host density increased. The present study can facilitate the optimization of laboratory conditions for the mass rearing of T. achaeae and present a starting point to enhance the biological control of several lepidopteran pest species.This study is the first to report the effect of temperature on two-sex life table parameters and functional response of


Enterocytozoon bieneusi is an intestinal pathogen that infects a wide range of species, including humans. Cattle constitute an important host for E. bieneusi; however, there is a scarcity of information on the prevalence and genotyping of E. bieneusi in cattle in the Hainan Province of China. In this study, PCR analysis of 314 fecal samples from cattle in six cities of Hainan was performed for genotype identification. The average prevalence of E. bieneusi in these animals was 9.9% (31/314), and ranged from 0.0% (0/12) to 20.5% (8/39). Five known genotypes – EbpC (n = 14), BEB4 (n = 12), J (n = 2), I (n = 1), and CHG5 (n = 1) – and a novel genotype: HNC-I (n = 1) – were identified. Genotypes EbpC and HNC-I were placed in zoonotic Group 1, and the remaining four genotypes  were placed in Group 2. Since 93.5% of the genotypes found in the cattle (29/31)  have previously been found in humans, these genotypes are probably involved in the transmission of microsporidiosis to humans. Enterocytozoon bieneusi, a zoonotic intestinal pathogen, infects a wide range of species worldwide [E. bieneusi through contaminated soil, feces, surfaces, water, as well as by improper farming practices, such as using untreated animal manure as fertilizer directly on open crops or tillage land [Enterocytozoon bieneusi has received considerable attention due to its known propensity to cause both water- and food-borne outbreaks of illness [orldwide , 24. Micage land . Enteroc illness .E. bieneusi genotypes from six groups, and more than 90% of human-pathogenic genotypes belonged to Group 1 or Group 2 [Sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene has revealed more than 500 genotypes  , 20, 54.E. bieneusi  and adults aged > 12 months (n = 296). Cattle were in good health at the time of sampling. Within 24 h of sampling, the labeled fecal bags were transported and stored in the laboratory at 4 °C and were processed within 48 h.In all, 314 fecal samples were gathered from 10 cattle farms in six cities of Hainan Province between March and December 2019 . The catg for 10 min. A QIAamp DNA stool mini kit  was used to isolate the genomic DNA of each processed specimen (approximately 200 mg), following the manufacturer’s instructions. A total of 200 mL AE elution buffer was used to elute the DNA, followed by storage at −20 °C before PCR analysis.All fecal specimens were filtered through sieve in distilled water, followed by centrifugation at 1500 ×Enterocytozoon bieneusi-specific nested primers and cycle parameters designed by Hamed Mirjalal were used to amplify a 410 bp sequence in the ITS region of the rRNA gene using TaKaRa Taq DNA Polymerase [lymerase . The PCRE. bieneusi-positive PCR products  was confirmed through bidirectional sequencing and the sequencing of additional PCR products. The Basic Local Alignment Search Tool (BLAST) and ClustalX 1.83 were used to compare the published GenBank sequences with the ones identified in this study to identify the genotypes of E. bieneusi. Genotypes that were identical to the genotypes deposited in the GenBank database were given the first published name, and those that generated ITS sequences with a single nucleotide substitution/deletion/insertion were identified as novel genotypes based on the DNA sequencing of minimum two PCR products [E. bieneusi was used for naming reference, following the established nomenclature system [The sequence accuracy of all products . The same system .A neighbor-joining phylogenetic tree was built using Mega X software, and the Kimura-2-parameter model with 1000 replicates to evaluate the relationship between the novel ITS genotype and the known genotypes, and to confirm the gene group designation.p-value < 0.05 was regarded as statistically significant.Fisher’s exact test and a Chi-square test were used to evaluate the difference in infection rates among different locations and ages, respectively, using SPSS v22.0 . A MT193626.The GenBank database accession number of the identified nucleotide sequence was E. bieneusi-positive, based on sequence analysis of the ITS region of the rRNA gene. A significant difference in the rate of occurrence of E. bieneusi was observed in cattle from the six cities (p < 0.05), with 20.5% (8/39) in Danzhou, 19.4% (6/31) in Wanning, 12.5% (12/96) in Lingshui, 4.9% (4/82) in Chengmai, 1.9% (1/54) in Haikou, and an absence of this parasite (0/12) in Ledong  were n Ledong .E. bieneusi in calves in Germany, there have been 38 published epidemiological reports on E. bieneusi conducted in 14 countries, and the average infection rates in these countries range from 2.0% to 21.4%  in young animals ≤ 12 months and 9.1% (27/296) in adult animals > 12 months. Although the infection rates in calves were higher than those in adults, the differences were not significant  , 7.7% (8/104), 10% (1/10), and 3.9% (13/332), respectively [E. bieneusi compared with heifers  and adults  [E. bieneusi in cattle, probably due to the underdeveloped immune systems of the young animals.Since the first report of to 21.4% . The inf.0–37.6% . This stn cattle . In the  > 0.05) . A studyectively . SimilarAF076042), with one base variation at position 237 (C → T). Out of the six genotypes, the most prevalent genotype was EbpC (14 specimens), which was found in four of the six locations, followed by BEB4 (12 specimens), but this genotype was only found in Wanning. Genotype J was found in two cattle from Chengmai. The remaining three genotypes I, CHG5, and HNC-I were found in a single specimen, with the former from Danzhou and the latter two from Wanning. These results differed from those reported from the other regions of China. For example, in Gansu, Guangdong, Henan, Ningxia, Jiangsu, Shaanxi, and Xinjiang provinces, genotypes J and I were reported to be the dominant genotypes, and in Heilongjiang, genotype O was dominant  and five known genotypes . The novel genotype showed high similarity to genotype EbpC . Genotype EbpC has been detected in humans, such as in cancer patients in Iran , in immu/31. GenoThe remaining genotype CHG5 and the novel genotype HNC-I were first identified in cattle here. Genotype CHG5 has been reported in goats with a wide distribution in China , 53. We E. bieneusi-infected cattle represent a potential threat to humans.The phylogenetic analysis revealed that EbpC and HNC-I, identified in this study, were divided into zoonotic Group 1, whereas genotypes BEB4, J, I, and CHG5 belong to Group 2 . In totaE. bieneusi in cattle from Hainan Province. Our results revealed a prevalence rate of 9.9% (31/314) for E. bieneusi within five of six cities in Hainan, China. We identified five known genotypes and a novel genotype. Genotype EbpC and novel genotype HNC-I were grouped into zoonotic Group 1, while genotypes BEB4, J, I and CHG5 were placed in Group 2. The observed high occurrence  of zoonotic genotypes  emphasizes the possible role of cattle in the transmission of E. bieneusi to humans, which requires further investigations to reduce the threats posed by these animals to public health.This study is the first evaluating the infection rates, genotype characteristics, and zoonotic potential of


Complicated acute biliary calculous disease poses clinical challenges. The European Society of Trauma and Emergency Surgery (ESTES) snapshot audit of complicated biliary calculous disease aims to make novel comparisons between self‐reported institutional adherence to the Tokyo guidelines (TG18) and ‘real‐world’ contemporary practice across Europe.A preplanned analysis of a prospective observational multicentre audit that captured patients undergoing emergency admission for complicated biliary calculous disease  between 1 and 31 October 2018 was performed. An anonymized survey was administered to participating sites.versus 38 per cent respectively; odds ratio 2·14 (95 per cent c.i. 1·37 to 3·35), P < 0·001). Six centres (24 per cent) self‐reported routinely performing blood cultures in acute cholecystitis; patient‐level audit data revealed that blood cultures were done in 47 of 154 patients (30·5 per cent). No centre self‐reported omitting antibiotics in the management of acute cholecystitis, and 144 of 154 (93·5 per cent) of patients in the snapshot audit received antibiotics during their index admission.Following an open call for participation, 25 centres from nine countries enrolled 338 patients. All centres completed the anonymized survey. Fifteen centres (60 per cent) self‐reported that a minority of patients were treated surgically on index admission, favouring interval cholecystectomy. This was replicated in the snapshot audit, in which 152 of 338 patients (45·0 per cent) underwent index admission cholecystectomy, 17 (5·0 per cent) had interval cholecystectomy, and the remaining 169 (50·0 per cent) had not undergone surgery by the end of the 60‐day follow‐up. Centres that employed a dedicated acute care surgery model of care were more likely to perform index admission cholecystectomy compared with a traditional general surgery ‘on call’ service  describe expert consensus for best practice in the management of complex acute biliary calculous disease. Self‐reported adherence (and limitations to this) were compared with ‘real‐world’ patient‐level data from a prospective observational study, the ESTES snapshot audit. Recognition of TG18 was high, but adherence to guidance on both index admission cholecystectomy and microbiological culture‐guided antibiotic therapy was poor.Low self‐reported and actual adherence to guidelines Acute complications of biliary calculi, such as complicated cholecystitis, choledocholithiasis with or without cholangitis, and biliary pancreatitis, commonly require urgent hospital admission for surgical careEfforts have been made to achieve expert consensus on the diagnosis and management of disease conditions caused by biliary calculi, in the form of the Tokyo guidelines, which were updated most recently in 2018 (TG18)The present authors hypothesized that, although regional and patient heterogeneity may account for some of the variability that could be expected in different clinical practices to treat these conditions, other causes such as unit policies and individual surgeon preference might also influence the treatment decisionsClinicalTrials.gov . The audit enrolled all consecutive patients admitted with complicated biliary calculous disease during the month of October 2018, and followed these patients for 60 days after admission (up to 31 December 2018). The database was closed for analysis on 1 February 2019. In May 2019, an anonymized follow‐up survey was completed by all 25 centres, assessing self‐reported awareness of and adherence to recommendations outlined in TG18. Survey respondents were asked to classify the model of unscheduled surgical care employed at their surgical department or hospital into one of the following categories: a dedicated acute care/emergency surgery service line  or a traditional ‘on call’ emergency service provided by general surgeons  with a primary commitment to elective surgical care.A prospective observational multicentre audit was conducted in line with a prespecified protocol that was registered with All adult patients (aged 18 years or over) admitted for acute calculous cholecystitis (American Association for the Surgery of Trauma (AAST) severity grade I–II or above), choledocholithiasis or complications of cholelithiasis and/or choledocholithiasis, or biliary pancreatitis were included in the study. Surgical procedures performed on these patients included cholecystectomy , choledochotomy/common bile duct (CBD) exploration (open or laparoscopic) or pancreatic necrosectomy. Data on endoscopic retrograde cholangiopancreatography (ERCP), radiological percutaneous cholecystostomy , percutaneous transhepatic drainage, stone removal or stent placement were also collected. Patients with uncomplicated biliary colic or biliary dyskinesia were excluded from the study.All participating centres had institutional review board approval or equivalent. No patient consent was sought as the study was purely observational and did not change the medical course of any patient. All data were deidentified at source when uploaded to the study database.https://www.project‐redcap.org/) without patient‐identifiable information. Centres were asked to validate that all eligible patients during the study period had been entered, and to attain more than 95 per cent completeness of data field entry before final submission. Quality assurance mentorship was provided by at least one consultant or attending‐level surgeon at every participating site.Data were recorded contemporaneously and stored on a secure user‐encrypted online platform  using R 3.6.0 El Capitan . Measures of central tendency are presented as mean(s.d.) (range) or median (i.q.r.) values, and comparisons were made with the χ2 test or ANOVA, as appropriate. An α significance level of 0·05 was used throughout.Descriptive and inferential statistical analyses were performed using Stata® 15.1  and the jamovi project version 1.2.16  in May 2018, 25 centres from nine countries  completed the local ethical approval process and proceeded to enrol patients prospectively. Fourteen (56 per cent) of the 25 centres described themselves as a university hospital or tertiary referral centre, and the remaining 11 (44 per cent) described themselves as a general/community hospital. The median catchment population of centres was 500 000 people. The majority of centres reported high volumes of eligible patients, with 22 (88 per cent) of the 25 centres performing more than 100 elective laparoscopic cholecystectomies per year. Similarly, 24 centres (96 per cent) reported receiving more than 300 admissions per annum for symptomatic biliary calculous disease.Table A dedicated acute care/emergency surgery service line  existed in seven (28 per cent) of the 25 centres, whereas general surgery on‐call managed and operated on patients in 18 (72 per cent) of the centres. Previous training in hepatopancreatobiliary (HPB) surgery had been undertaken by surgeons in eight (32 per cent) of the 25 centres Table 1.versus 46·2 per cent). The mean(s.d.) age of patients at the time of diagnosis was 64·5(18·4) years. The mean(s.d.) BMI was calculated as 28·5(6·4) kg/m2. Some 16·3 per cent of the patients were current smokers of tobacco products, 34·9 per cent were ex‐smokers (stopped smoking more than 6 weeks before admission), and 48·8 per cent had never smoked. The mean(s.d.) age‐adjusted Charlson Co‐morbidity Index score was 6·26(4·1), and the mean(s.d.) APACHE‐II score was 12·3(7·6) . Women outnumbered men (53·8 6) Table 2.Table Acute calculous cholecystitis was present in 154 (45·6 per cent) of the 338 patients, acute biliary pancreatitis in 71 (210 per cent) and choledocholithiasis in 108 (32·0 per cent), of whom 47 (43·5 per cent) had cholangitis. Five patients (1·5 per cent) were admitted for treatment of Mirizzi syndrome or bilioenteric fistula Table 2.The Tokyo guidelines (TG18) set out a number of best‐practice parameters deemed by expert consensus to contribute to optimal care. Self‐reported (retrospective) adherence to the guidelines was assessed, along with prospective data on adherence from the ESTES snapshot audit of the same centres.Table P < 0·001).Survey respondents were asked to estimate the percentage of patients in their practice who underwent cholecystectomy during the index hospital stay. Fifteen (60 per cent) of the 25 centres self‐reported that a minority of patients with acute cholecystitis had cholecystectomy on the index admission, favouring interval cholecystectomy. When polled on reasons for this practice (multiple answers were permitted), acute care surgeons solely cited lack of emergency operating room access, while the responses from general surgery centres were more diverse Table 3. Centrversus 38·4 per cent respectively; OR 2·14 (95 per cent c.i. 1·37 to 3·35), P < 0·001)  Table 3.a priori decision to perform laparoscopic cholecystectomy on index admission: .Previous training in HPB surgery did not significantly influence the declared Of the 338 patients enrolled in the study, 169 (50·0 per cent) had a surgical intervention and 169 had not undergone surgery by the end of the 60‐day follow‐up period. Of patients who had a cholecystectomy, 152 (89·9 per cent) had the operation during the index admission, and only 17 (10·1 per cent) were reported as having been operated on after discharge from the index admission but before closure of the study database. The median interval from index admission to cholecystectomy was 66 (i.q.r. 43–71) days.Table P = 0·944).Survey respondents were asked whether, in their practice, admission blood and intraoperative bile cultures were performed for patients admitted with acute cholecystitis. Six (24 per cent) of the 25 centres stated that they routinely performed blood cultures, and six (24 per cent) reported routinely performing intraoperative bile cultures during cholecystectomy. When patient‐level data from the snapshot audit of the same centres were reviewed, 47 (30·5 per cent) of 154 patients with acute cholecystitis had blood cultures drawn, and 35 (22·7 per cent) had intraoperative bile cultures sent for microbiological analysis Table 4. ReporTable P = 0·717). Eleven centres (44 per cent) elected not to commence antibiotics in patients with biliary pancreatitis. Of the 14 centres that did commence empirical antibiotics, piperacillin–tazobactam was the favoured drug , with meropenem and co‐amoxiclav each favoured in three centres, and cephalosporin plus metronidazole combination in one centre.Survey respondents were also asked whether, in their practice, admission blood cultures were performed for patients admitted with acute biliary pancreatitis. Eight (32 per cent) of the 25 centres stated that they routinely performed blood cultures on admission. When patient‐level data from the snapshot audit of the same centres were reviewed, 28 per cent (20 of 71) of patients with acute pancreatitis had blood cultures drawn for microbiological analysis Table 5. ReporTable Addressing the question of the therapeutic sequence for the management of choledocholithiasis in patients fit for cholecystectomy, 20 (80 per cent) of the 25 centres reported that they favoured a staged approach, with upfront ERCP followed by cholecystectomy . A minority of survey respondents favoured simultaneous cholecystectomy and either operative CBD exploration  or rendezvous intraoperative ERCP  as a one‐stage procedure Table 6.Incremental improvements in patient outcomes for certain common surgical conditions are achievable by the standardization of patient care through practice management guidelinesThe Tokyo guideline TG18 has been promulgated widely, and its apparent success may be measured by how it is now part of the vernacular whenever the management of complicated calculous biliary disease is being discussedIn the present study, self‐report measures of TG18 compliance among surgical services providing acute surgical care in 25 hospitals across nine countries were compared with real‐world prospective data by snapshot audit from the same centres. The survey asked respondents to report their practice in various domains of TG18, without explicitly referencing these guidelines.TG18 advocates definitive surgical intervention  on the index admission in patients with acute cholecystitis or biliary pancreatitis, citing both prospective longitudinal studies and retrospective observational studies that demonstrated a significantly lower incidence of disease recurrence, hospital readmission, and overall disease‐specific complications when the procedure was performed within 7 days of the onset of symptoms in the appropriate patient (recommending within 72 h as preferable)P = 0·013).When asked why index admission cholecystectomy was not performed more frequently in their centre, despite TG18 best practice, respondents cited lack of timely access to an emergency operating room, delayed patient presentation, local surgical culture, or surgeon preference for interval cholecystectomy. Although the effect of unscheduled admissions on hospital bed capacity and the knock‐on ability of a centre to deliver elective surgical care was beyond the scope of this study, it may reasonably be presumed that uncoupling elective and emergency service lines and resource allocation would facilitate and incentivize definitive care of index admissions. Indeed, respondents working in centres employing a resourced dedicated acute care surgery service line were significantly less likely to cite surgeon preference as the reason for poor rates of index admission cholecystectomy ; P. Healy, C. Moriarty, C. Power, N. Knightly, A. D. K. Hill ; D. C. Winter, M. E. Kelly, B. E. Creavin, É. J. Ryan, C. C. Duffy ; M. Sugrue, M. H. Moore, L. Flanagan ; J. Ryan, C. Keady, B. Fahey, K. L. McKevitt, K. Barry ; K. C. Conlon, K. Mentor, A. Kazemi‐Nava, B. Julies ; P. F. Ridgway, D. O. Kavanagh, M. Whelan, M. Donnelly, C. McCarrick, U. Muhammad, T. M. Connolly, P. C. Neary ; S. Magalina, V. Cozza, A. LaGreca, D. Gui ; A. Malagnino, M. Zago, M. Montuori ; A. Biloslavo, N. Samardzic, S. Fracon, D. Cosola, N. de Manzini ; U. Fernandes, P. Avelar, R. Marques, A.S. Esteves, A. Marçal, C. Gomes ; D. Machado, T. Teles, S. Neves, M. Semiao, R. Cunha ; J. Pereira, J. Constantino, M. Sá, C. Casimiro ; L. Ionescu, R. Livadariu, L. Stirbu, R. Danila, D. Timofte, B. Astefaniei ; A. Landaluce Olavarria, B. Estraviz Mateos, J. Gonzalez Taranco, D. Gomez, J. Barrutia, J. Zeballos ; D. Morales Garcia, A. Lozano Najera, E. Gonzalez Tolaretxipi ; L. Tallon‐Aguilar, J. Pintor‐Tortolero, A. Sanchez‐Arteaga, V. Duran‐Muñóz Cruzado, V. Camacho‐Marente, J. Tinoco‐Gonzalez ; A. Älverdal ; S. Redeen ; A. Mohammad, R. Ahl, M. Wikström ; S. Marinos, N. Warner, R. Patel, T. Magro, R. Sunthareswaran ; A. Mihailescu, G. Pokusewski, A. L. Bubuianu ; C. Dimitriu, M. Paraoan ; A. Desai, K. Jones, M. Mlotshwa, K. Ross, S. Lambracos, Y. Tryliskyy ; D. C. Cullinane .The authors declare no conflict of interest.