Wave interference between these components limits the accuracy of DPOAEs when evaluating the function of the cochlea with conventional continuous stimulus tones. Here, DPOAE components are separated in the time domain from DPOAE signals elicited with short stimulus pulses. The extracted nonlinear-distortion components are used to derive estimated distortion-product thresholds (EDPTs) from semi-logarithmic input-output (I/O) functions for 20 normal-hearing and 21 hearing-impaired subjects. I/O functions were measured with frequency-specific stimulus levels at eight frequencies f2 = 1,…, 8 kHz (f2/f1 = 1.2). For comparison, DPOAEs were also elicited with continuous primary tones. Both acquisition paradigms yielded EDPTs, which significantly correlated with behavioral thresholds (p < 0.001) and enabled derivation of estimated hearing thresholds (EHTs) from EDPTs using a linear regression relationship. DPOAE-component separation in the time domain significantly reduced the standard deviation of EHTs compared to that derived from continuous DPOAEs (p < 0.01). In conclusion, using frequency-specific stimulus levels and DPOAE-component separation increases the reliability of DPOAE I/O functions for assessing cochlear function and estimating behavioral thresholds.Distortion-product otoacoustic emissions (DPOAEs) arise in the cochlea in response to two tones with frequencies  I.The healthy cochlea amplifies sound by actively  enhancinf1 and f2 where f2 > f1  with frequencies f1 and f2. Because of its nonlinear dependence on stereocilia deflection, the receptor current exhibits intermodulation products, which are coupled into the cochlear fluid as vibrations by mechanical forces from the electromechanical transducer of the OHC soma  which, by definition, is produced when stimulating simultaneously with two tones, with frequencies denoted by membrane . The firomponent , referre2, of the second stimulus tone, called the DPOAE input-output (I/O) function, to obtain DPOAEs at low stimulus levels. This approach has been shown to enhance the sensitivity of DPOAEs for detecting cochlear damage and to increase their correlation with auditory thresholds  by extrapolating the linear regression line to the abscissa  . Similarf2 stimulus and analyzing the DPOAE signal in the time domain . In order to identify hearing-impaired ears, behavioral thresholds (BTs) were recorded with clinical pure-tone audiometry . Subjects were classified as normal-hearing if all BTs for frequencies between 1 and 8 kHz were better than 20 dB hearing level (HL). BTs for hearing-impaired ears ranged from 0 to 77 dB HL with an average value of 24 ± 18 dB HL . All subjects were free of any conductive hearing impairment as ascertained by standard 226-Hz tympanometry  and otoscopy. Measurements of clinical, notched-noise, auditory-brainstem responses (ABR) for 1, 2, and 4 kHz and stimulus levels from 25 to 75 dB nHL in 10-dB steps  and acoustic reflex measurements at 0.5, 1, 2, and 4 kHz (Madsen-Zodiac 901) were used to exclude possible severe neural conditions for the hearing-impaired group. Subjects were included only if ABR waves V were detectable for at least one of the investigated frequencies. To avoid false-positive exclusions in cases without identifiable ABR signals, subjects were also included if at least one ipsilateral stapedius reflex could be detected. In 17 hearing-impaired subjects, ABR-wave V was detectable for at least one test frequency for stimulus levels equal to or below 65 dB nHL. In the remaining four subjects, ipsilateral stapedius reflexes were detectable at two or more frequencies. The subjects had no history of tinnitus.DPOAE I/O functions were recorded unilaterally from 20 normal-hearing and 21 hearing-impaired subjects with sensorineural hearing loss. Subjects were between 18 and 70 years and the normal-hearing group was significantly younger (mean age: 27.6 ± 4.2 years) compared to the hearing-loss subjects .OAE measurements and Békésy audiometry were performed unilaterally using an ER-10 C DPOAE probe-microphone system  connected to a 16-bit analog output card and a 24-bit signal acquisition card  situated in a commercially available PC. The sampling frequency was 102.4 kHz. Stimulus generation and data acquisition were controlled by a custom-built toolbox implemented in LabVIEW . The sound pressure of the ER-10 C speakers was ascertained by in-ear calibration, which was repeated every 120 to 240 s depending on the acquisition progress. Both the output of the speakers and the recorded microphone signal were corrected for the transfer functions of an artificial ear simulator  and of the ER-10 C microphone to yield DPOAEs, which are considered to correspond to recordings close to the tympanic membrane. Further details of the calibration routine are given elsewhere  neighboring frequencies, in order to account for the frequency-dependent bandwidth of the short-pulse f2 stimulus. The frequency range spanned by the lowest and highest neighboring frequencies was 80 Hz at f2 = 1 kHz and increased to 480 Hz at f2 = 8 kHz. These frequency ranges were similar to the bandwidths of the associated f2 pulses.A modified method of Békésy tracking audiometry was performed using the ER-10 C ear probe to assess behavioral thresholds in each subject directly before the OAE data acquisition started. The sound pressure of the continuous tone was controlled by the data acquisition software while the subject was required to indicate perception of the stimulus by pressing or releasing a button. The output level, L, started at −20 dB sound pressure level (SPL), well below hearing threshold, and increased in 0.1-dB steps with an alteration rate of 8 dB/s. The acquisition setup gradually decreased the intensity-rate change to avoid clicks in the presentation of tones with high output level . The subject was instructed to press and hold down a button if the sound was perceived, thereby establishing an upper pure-tone threshold. While the button was held down, the system decreased the output level until the subject lost perception of the sound. Releasing the button indicated a lower pure-tone threshold. The mean value of the lower and upper threshold provided an estimate of the auditory threshold. On average, the elapsed time between the detection of these two thresholds was 2.25 ± 0.79 s. The maximum output level was set to 85 dB SPL. As in f2 and its neighboring frequencies, the median values and the standard deviations were computed separately for the lower and upper thresholds across the three Békésy recordings. A behavioral threshold which differed from a lower- or upper-threshold median by more than three times the associated standard deviation was classified as an outlier and replaced by the median value of the lower or upper threshold of the frequency group. This procedure enabled frequency-specific outlier correction even for hearing-impaired subjects. Finally, the estimates of the behavioral thresholds at the f2 frequencies, denoted as LBT, were computed by averaging across frequencies for each frequency group, in order to mimic the spectral spread of the pulsed DPOAE stimuli.Three successive Békésy measurements were recorded and averaged to obtain a reliable estimate of the behavioral threshold. To reduce the impact of outliers, a correction algorithm similar to the one introduced in D.f2 ≤ 8 kHz with a constant frequency ratio of f2/f1 = 1.2. L2 values ranged from 25 to 75 dB SPL in 5-dB steps with frequency-dependent L1 values representing preliminary results based on a subset of a recently published study  filter with an order of 1200 and filter coefficients computed using a Hamming window. The filter bandwidths were defined asf2 stimulus. The maximum values were For each stimulus pair, the corresponding DPOAE signal, The SNR for the short-pulse DPOAE measurements was defined by the ratio of the amplitude of the extracted nonlinear-distortion component, 2.fDP. This yielded DPOAEs which represent the vector sum of the nonlinear-distortion component and the coherent-reflection component , particularly at moderate levels, which were evident for both continuous and short-pulse stimulation. Therefore, a correction algorithm was implemented, similar to the f2, let N be the number of stimulus levels for which the 10-dB SNR criterion was satisfied   and, consequently, only one type was assigned to an I/O function.Denoting the amplitudes of the nonlinear-distortion and coherent-reflection components by III.A.a, decreased from 274 to 237 (86.5%) in the case of short-pulse DPOAEs and from 302 to 238 (78.8%) for continuous DPOAEs; that is, these acceptance criteria resulted in a greater proportion of the continuous DPOAEs being rejected. However, incorporating the HLC algorithm . None of the evaluation parameters exhibited significant differences between acquisition paradigms for the unmodified I/O functions , nor when corrected for high-level deviations from the expected straight-line behavior .Table B.2 ≥ 45 dB SPL (Sec. F (gray bars), of the distribution of the relative occurrence, m, of FS-affected I/O functions across subjects. For each subject, m was computed as the number of FS-affected I/O functions divided by the number of computable I/O functions. The histogram indicates that the proportion of I/O functions with an interfering coherent-reflection component tends to be uniformly distributed across subjects. The empirical distribution function, Fc [dashed line in Fig. Fc(m) = 0.585 for m = 0.5, implying that in 41.5% of the subjects more than half of the computable I/O functions are FS-affected. There was no correlation between the ratio BT . The portion of FS-affected I/O functions was similar for normal-hearing (LBT < 20 dB HL) and hearing-impaired thresholds with 46.4% (109/235) and 46.2% (18/39), respectively, suggesting that an interfering coherent-reflection component may also occur in hearing-impaired subjects.According to PBF decomposition of the short-pulse DPOAE responses at a level LSPL Sec. , 46.4% ((t) Fig. . The assp = 0.03), with corresponding interquartile ranges (IQR) of 0.06 and 0.12. For the continuous DPOAEs, 38.6% of the I/O functions exhibited p = 0.396) and the proportion of I/O functions with The impact of a pronounced coherent-reflection component on the growth behavior and shape of the I/O functions was quantified with 2 = 45 dB SPL using PBF decomposition  than EDPTs based on continuous DPOAEs (p < 0.001). Regression analysis between LBT and LEDPT reveals a linear relationship, enabling estimated hearing thresholds (EHT), LEHT, to be derived from EDPTs according toFor both acquisition paradigms, EDPTs were related to behavioral thresholds (BTs) estimated by the adapted version of Békésy tracking audiometry Sec. . Figure lse Fig.  and contlse Fig.  stimuli.p < 0.01). To estimate the impact of two-component interference on the accuracy of p = 0.24; Figs. p = 0.02), which is also evidenced by the lower standard deviation, The accuracy of the auditory-threshold estimation procedure was assessed using the standard deviation, ta Table . Figure lse Fig.  and contlse Fig.  stimulatAEs Fig.  comparedAEs Fig. .a and columns labelled HLC) results mainly from the lower acceptance rates at f2 = 1 and 8 kHz of only 65.9% and 46.4%, respectively  and 71.7% (33/46), respectively, but declines notably at thresholds above 40 dB HL to 18.9% (7/37) for the continuous data and 5.4% (2/37) for the short-pulse data. Figure 74.7% (245/328) of all thresholds measured with Békésy audiometry were below 20 dB HL Fig. . I/O funD.f2 yields an objectively measured audiogram for each subject. Figure p = 0.006). Despite using the HLC algorithm, continuous DPOAEs remained prone to large deviations due to two-component interference—they result in maximum deviations, p = 0.003).Exploiting the linear relationship between BTs and EDPTs enables estimation of hearing thresholds using Eq. cf. Fig. , whereasIV.DPOAE I/O functions based on the extracted nonlinear-distortion components enable the estimation of auditory thresholds with high accuracy and, therefore, offer a promising approach for objectively assessing hearing status. Section A.Short-pulse stimulation enabled the separation of the two DPOAE components by means of onset decomposition (OD). The fidelity of the separation can be directly assessed in DPOAE responses with destructive interference, where both components become readily distinguishable in the time signal Fig.  and in tf2 = 1.5,…, 2.5 kHz. That study employed a pre-defined sampling instant between 8 to 10 ms relative to the f2 onset. However, the optimal sampling instant for OD was found to decrease with increasing stimulus level. This finding is in accordance with other studies showing that latencies of DPOAEs vary considerably with stimulus frequency and level  in the time domain by fitting the DPOAE short-pulse response to a mathematical model that mimics the superposition of the components . Both methods provide a generally reliable extraction of the nonlinear-distortion component, as supported by the almost complete removal of fine structure. OD slightly underestimated the amplitude of the nonlinear-distortion component because it samples the DPOAE signal prior to its maximum. PBF decomposition resulted in extracted components, which reproduced known properties of the two DPOAE components reported by others  fitting the data with different slopes depending on the DPOAE growth behavior  behavior , is alsoC.f2 ≤ 1 kHz and the higher number of hearing-impaired subjects than in the present study. Furthermore, for both acquisition paradigms, the HLC algorithm used in the present study appears to be another beneficial factor for the acceptance rate. While the acceptance rate for the corrected continuous DPOAE data was larger than the 84.7% reported by f2 ≤ 3 kHz and only from the normal-hearing population . For this subset, the acceptance rate for I/O functions recorded with short-pulse stimulation increases to 97.8% (90/92), which is close to the results of The number of DPOAE I/O functions complying with the objective evaluation criteria Sec. , definedD.f2 = 1 to 8 kHz with hitherto unreported accuracy of f2, the coherent-reflection component may induce errors in the threshold estimate. Therefore, extracting the nonlinear-distortion component from short-pulse DPOAE recordings, as was done in this study using OD, can be one reason for the increased accuracy. This suggestion is supported by the significantly smaller value of EDPT < −10 dB SPL; such I/O functions were excluded from further analysis. Nonetheless, when using the continuous data, the present study also achieved a smaller estimation error than in those earlier studies. The difference might be related to the investigated population, which in the present study included fewer subjects with profound hearing loss than in previous studies. Since DPOAEs cannot assess the functional state of the auditory system beyond the cochlear amplifier, such as the inner hair cells (IHCs) or the auditory nerve, the observed EDPTs might underestimate BTs in cases of severe hearing loss. However, in the present study, only 11.3% of BTs exceeded 40 dB HL. Furthermore, a large number of hearing-impaired subjects would primarily yield an increased number of incomputable I/O functions due to insufficient SNR, as in the study of Both DPOAE acquisition paradigms yielded EDPTs, which allowed the prediction of behavioral thresholds in a clinically relevant frequency range from ion Sec. . These vB) Table . Furtherf2-tonotopic place on the basilar membrane [Eq. f2 ≤ 3 kHz (except f2 = 2 kHz) than reported by Moreover, the present study utilized optimized primary-tone levels to maximize the DPOAE amplitude by accounting for the different compressional behavior of the stimulus traveling waves at the rane Eq. . Using of2 and neighboring frequencies were performed using a modified form of Békésy tracking audiometry (Sec. f2 eliminated the fine structure in auditory thresholds. Additionally, this procedure enabled the correction of BTs for outliers and also the computation of standard deviation as a measure of reproducibility among the three Békésy measurements. The median value of the standard deviation of all BTs across frequencies and subjects was 2.37 dB, which was larger than the average standard deviation of 1.38 dB in the study of When relating DPOAEs to hearing status, acquisition of the behavioral threshold bears additional measurement uncertainties. In the present work, to increase the accuracy of BT estimates, three consecutive measurements at try Sec. . The accEHT when using only the nonlinear-distortion component in the regression analyses. This improvement is in accordance with previous results of f2 ≤ 2.5 kHz, and reported a f2 ≤ 3 kHz, and LBT < 20 dB HL, the improvement is still not as large: Whereas f2 = 1 and 8 kHz (Table f2 = 1.5 kHz (The low correlation between BTs and EDPTs at Hz Table  resultedon Table , except obe Sec.  may haveobe Sec. . HoweverE.Despite being related to the BT, the EDPT only provides a metric for the functional state of the cochlear amplifier, or in other words, only for the pre-neural component of cochlear function up to deflection of the IHC stereocilia. Inter-subject variation in the neural system with regard to action-potential generation, neural transmission, and other possible sources of interference must, by definition, influence the correlation between BTs and EDPTs. The significance of these influences can be gauged with the aid of a simple model presented by 2 for The median value ata Fig. . In abseIn summary, this analysis leads to two conclusions. First, for short-pulse DPOAEs, the error associated with diagnosing the state of the cochlear amplifier with this method has a standard deviation below 5 dB. Second, there is no evidence for an increase in the variance of the data with increasing hearing loss cf. Fig. . Thus, fF.2 levels cannot evoke a DPOAE with suitable SNR. By extending the acquisition software to enable the selection of stimulus levels adaptively according to the SNR of the acquired DPOAEs, it should be possible to reduce the acquisition time to well below 5 min for short-pulse stimulation, feasible for daily clinical routine.The present data suggest that for a population with normal hearing or mild-to-moderate hearing loss, short-pulse DPOAE I/O functions enable accurate estimation of behavioral thresholds, not only for the pooled data but also for individual subjects. In the case of continuous stimulation, interference of the DPOAE components leads to significantly larger errors in the individual objective audiogram. The present work provides only a limited statement about the measurement time of short-pulse acquisition necessary in clinical routine, because identical stimulus levels were used for both the normal-hearing and the hearing-impaired group. Furthermore, the 10-dB SNR criterion for the short-pulse multi-frequency acquisition was restricted to the DPOAE with the lowest SNR within each multi-frequency acquisition sequence, causing averaging times for the remaining DPOAEs in the same sequence to be longer than necessary. On average, the measurement time to obtain threshold estimates for all eight frequencies was 16.45 ± 1.65 min and 6.85 ± 2.76 min per subject for short-pulse and continuous stimulation, respectively. These measurement times include the acquisition of eleven DPOAEs per I/O function. In the case of normal-hearing subjects, this procedure leads to oversampling of the I/O function, while for hearing-impaired subjects a large number of the LV.Both DPOAE acquisition paradigms, incorporating either short-pulse stimuli or continuous primary tones, yield estimates of behavioral thresholds with high accuracy, supporting the use of frequency-specific stimulus levels and the high-level correction of semi-logarithmic I/O functions for deviations from the expected linear shape. Onset decomposition successfully extracts the nonlinear-distortion component from short-pulse DPOAE recordings. Utilizing I/O functions solely based on the extracted nonlinear-distortion components significantly improves auditory-threshold estimation for normal-hearing subjects and patients with mild-to-moderate hearing loss induced by an impaired cochlear amplifier. The high correlation of the EDPTs with behavioral thresholds demonstrates that individual audiograms representing the state of the hearing path up to the IHC stereocilia can be acquired with high reliability.


To this end, all workers in the refinery were chosen. In this research, the effects of history of disease and injury including trauma, electric shock, meningitis-typhoid disease and genetic illness as well as contact with lead, mercury, CO Specifications TableValue of the data•Data describe those factors which affect the acceleration of hearing reduction among which are useful for promoting the workers' education in order to prevent this complication.•Due to the importance of the relationship between hearing reduction with history of diseases and injuries among petroleum refinery workers, these factors are discussed in this article.•The results showed that hearing reduction can be decreases the efficiency of workers.•The results of this study can be used to develop a prevention program to decrease hearing reduction among the workers of petroleum refinery.•Results are also important for workers with hearing reduction especially those workers who were detected in this study.1The dataset of this article provides information on the relationships between hearing reduction with history of disease and injuries among workers in Abadan Petroleum Refinery, Iran. 22.1Petroleum Refinery of Abadan was selected for the purpose of the study. Abadan with a population of 300,000 people is one of the metropolitan cities of Khuzestan province 2.22 and alcohol consumption 291 workers of Abadan petroleum refinery participated in this study. A questionnaire was constructed by the researcher based on the risk factors adopted from Williams’ Obstetrics and Gynecology (based on the risk factors adopted from Williams’ Obstetrics and Gynecology),which comprised the demographic data  and questions related to the causes and effective factors of the acceleration of hearing reduction, which included history of some diseases and injuries , contact with lead, mercury, CO


Roscoea, like other members of ginger family, exhibits the combination of floral traits that would fit pollination by long distant foragers such as bees, birds or flies. We studied the pollination biology of Roscoea alpina, observed potential floral visitors and determined their foraging behaviour, visitation frequency and pollination efficiency, to seek evidence in support of the pollination syndrome hypothesis. We also measured the floral spectra of R. alpina flowers to evaluate if signals fit with the currently known framework for observed floral visitors. We found that R. alpina have autonomous selfing and pollinator-mediated crossing, but lack apomixis. We observed that a beetle (Mylabris sp.), and a moth (Macroglossum nycteris) visit the flowers of R. alpina for pollen and nectar feeding respectively. Our field observations, the stigmatic pollen count and fruit set data indicated that the visit by the beetle was legitimate, while that of the moth was illegitimate. Emasculated flowers visited by beetles set as many fruits and seeds/fruit as auto-selfed and naturally pollinated flowers, while emasculated flowers excluded from beetle visits did not set fruit and seed; indicating that a single visit of a beetle to the flowers of R. alpina can facilitate pollination. We found that flower spectral signal of R. alpina does not fit typical spectra previously reported for beetle or bee-visited flowers. Our results suggest that, to ensure reproductive success in alpine habitat, R. alpina has evolved autonomous selfing as a predominant mode of reproduction, while beetle pollination would promote genetic diversity of this plant species. The visitation of beetles to the flowers of R. alpina, despite floral signal mismatch with the classically associated beetle vision, suggests that a different visual processing may operate in this plant-pollinator interaction at high altitudes.The Himalayan endemic alpine genus  Plant-pollinator interactions form complex relationships that have been intensively studied for over a century to understand the evolution of angiosperms –7. EvideGingers (family Zingiberaceae) with about 1300 species in 52 genera are one of the earliest angiosperms that evolved in the Cretaceous with a broad tropical distribution ,21. FollRoscoea with 22 species is a Himalayan endemic alpine genus of the predominately tropical family Zingiberaceae [Roscoea relative to other members of the family is the consequences of the uplift of the Himalayas subsequent to the collision of the Indian plate with Eurasian plate [Roscoea contain nectar as a reward, indicating pollination by nectar feeders such as bees, birds, flies, moths etc. [Roscoea such as wide labellum, long corolla tube with nectar, flexible anther, nectar source located away from the reproductive organs, and absence of fragrance suggests adaptation toward long-tongued insects for pollination success [R. purpurea [beraceae ,25. The an plate . Like otths etc. . Moreove success ,27. Thispurpurea ,19,28,29R. alpina, to test for potential evidence supporting a pollination syndrome. We hypothesized that consistent to the closely related R. purpurea [R. alpina might involve highly specialized mutualism with long-tongued insects to enable pollination. Surprisingly, however, our observations revealed that a beetle (Mylabris sp.) and a moth (Macroglossum nycteris) were the only observed floral visitors of R. alpina. We subsequently focused on the following questions: (i) How does pollination occur in R. alpina? (ii) Do the observed visitors (beetle and/or moth) act as the effective pollinator of R. alpina? (iii) How might the floral spectral signal of R. alpina fit with the currently known framework for beetle and moth vision?In the current study, we investigated the pollination biology of purpurea , R. alpiWe obtained general permission from local community forestry users groups, local governmental bodies (Village Development Committee) and Annapurna Conservation Area Project to conduct the research. None of our model species were regarded as within a threat category, and ethics is not required for research of insect observational studies.Roscoea alpina is a true alpine species within the genus and is distributed between the elevations of 2130 m to 4270 m in the Himalayan regions  [Rhododendron, Pinus, and/or Betula forest. It is perhaps the smallest species amongst the Himalayan Roscoea. The erect pseudostem that grows out from the underground rhizome may produce up to four linear, broadly elliptic or lanceolate leaves. The first leaf is slightly auriculate and widest at the base while the rest of the leaves are widest at the middle. Flowering occurs from the end of May to the end of July. R. alpina produces single inflorescence and only one flower of the inflorescence open at a given time, although an inflorescence bears up to 5 flowers. Flowers are without exerted peduncle and have previously reported pinkish to white flowers, colour characteristics, considering human perception [he east) . It growrception .R. alpina at three different sites; Lete , Mustang; Poonhill , Myagdi; and Chheplung , Solukhumbu, Nepal  and snowfall in winter (November-February) (Pers. obs BRP). The temperature of Poonhill ranges from -3° to 18°c in summer time [We studied the floral biology and observed the floral visitors of u, Nepal . The vegmer time .R. alpina at three sites  were measured from the freshly opened flowers (n = 20 for each trait) following the method of Paudel et al. [R. alpina vary spatially, we analyzed the differences in floral traits among the three sites using a one-way ANOVA. To find the stigma receptivity, we used Dimethylthiazol-diphenyl-tetrazolium bromide (MTT) to test for the presence of dehydrogenase on the stigma following the method used by Rodriguez-Riano and Dafni [We recorded the floral biology of ee sites , for twol et al. . To testnd Dafni , Wang etnd Dafni  and Fan nd Dafni . If the nd Dafni –33.R. alpina (at Poonhill) to provide initial insights into whether flower spectra might play a role in signal evolution for this species. Specifically, recent work [R. alpina. We used an OCEAN OPTICS spectrophotometer  with a PX-2 pulsed xenon light source attached to a computer running SPECTRA SUITE software and measured multiple individuals flowers from 300–700 nm wavelength following methods described in Chittka and Menzel [To understand plant-pollinator interactions, it is important to consider the different visual capabilities of various flower visitors and reliable measurements of flower spectra . We thusent work  shows thd Menzel , Dyer etd Menzel  and Shred Menzel . We usedR. alpina at three sites across central Nepal  at the distance of more than 100 m and observed all floral visitors of R. alpina within the plots. Foraging behaviour of the visitors (legitimate or illegitimate) was recorded by direct observation. The visit was considered legitimate if the visitor’s body touched the reproductive parts of a flower. We estimated the visitation frequencies of the floral visitors in terms of numbers of visit flower-1hour-1. To test if visitation frequencies differ among the plots and between the years, we analyzed the data using two way ANOVA with plots and years as fixed factors. Finally, visitation frequencies from three independent plots were pooled together and average values of two years were computed. The voucher specimens (plant samples and beetles) were deposited at Annapurna Natural History Museum and Herbarium Center, Prithvi Narayan Campus, Tribhuvan University, Nepal.We observed the floral visitors of al Nepal , from eaR. alpina at Chheplung and Lete, thus we were unable to conduct manipulated pollination experiments at these two sites. Hence, we conducted the manipulated pollination treatments only at Poonhill  Natural pollination- flowers were tagged and left untouched; (ii) Autonomous selfing- flowers were covered by mesh bags to exclude the pollinator; (iii) Apomixis- flowers were emasculated and covered by fine mesh bags to exclude the pollinators; (iv) Pollinator mediated crossing (emasculated and open)—flowers were emasculated (cleared off the self-pollens by gentle brushing) in the early morning on the first day of anthesis before anther dehisces following the method outlined in Paudel et al.[Among the three sites selected for the current study, there was low density of flowering individuals of Poonhill . To undeel et al. and leftR. alpina, we analyzed the difference in fruit set percentage and seed number per fruit between autonomous selfing and natural pollination. Similarly, to evaluate the contribution of pollinators for the natural breeding of R. alpina, we computed the differences in fruit set proportion and seed number per fruit between natural pollination and emasculated-open pollination (pollinator mediated crossing). To test for self-compatibility in R. alpina, we analyzed the fruit set and seed set differences between hand self-pollinated and hand cross- pollinated flowers. For all these analyses, we used generalized linear models (GLMs) with binary distribution of errors for analyzing the differences in fruit set percentages while we used GLMs with Poisson distribution of errors to estimate the differences in seed number per fruit. We used factorial GLM to test if fruit set and seed set are affected by treatment and year factors.To quantify the capacity of autonomous selfing and its contribution for the natural breeding system of R. alpina, we covered the matured buds (n = 240) with fine mesh bags until anthesis. Upon anthesis, flowers were emasculated in the early morning before pollinators start to visit the flowers. Those experimental flowers were assigned into three groups (n = 80 for each group), in a counterbalanced random fashion. (1) and (2) Emasculation and exposure to the pollinators. When a pollinator (either a beetle or a moth) visited the experimental flower assigned to either group 1 or group 2, it was freely allowed to forage. Subsequent to the departure of the pollinator from the flower, the stigma was immediately collected and fixed in 70% ethanol, if the flower was assigned to group 1 while the flower was immediately covered by a fine mesh bag until wilting, if it was assigned in group 2. (3) Emasculation and pollinators exclusion- emasculated flowers were left covered by fine mesh bags until wilting. This treatment was designed to exclude the potential pollinators and to check if emasculated flowers set fruit/seed without a pollinator visit. Fruit set by treatments (2) and (3) were collected after 20 days, and percentage of fruit set and seed number per fruit were quantified. Emasculated flowers visited by the moth (n = 40) neither set fruit and seed nor received any pollen grains on their stigma. We repeated the experiment for two consecutive years and found the same result. Thus, we concluded that moths were not a viable pollinator of R. alpina, and thus moths were not considered for subsequent analysis (see below). The potential importance of a beetle to the flower of R. alpina was assessed by analyzing the difference in fruit set percentage and seed number per fruit between emasculated-open flowers visited by the beetles (treatment 2) and emasculated flowers that were excluded from the beetle’s visit (treatment 3). We also analyzed the differences in fruit set percentage and seed number per fruit of emasculated-beetle pollinated flowers with natural pollination and autonomous selfing to estimate the potential efficiency of beetle, using GLM with binary and Poisson distribution of errors respectively.To measure the potential importance and pollination efficiency of a visitor to the flowers of R. alpina by a beetle during a single foraging bout (treatment 1) was counted under the microscope adopting the method outlined in Dafni et al. [R. alpina, we used the formula; PEI = visitation frequency × stigmatic pollen deposition following Ne’eman et al.[t test to analyze the differences in PEI of a beetle between two years.The number of pollen grains deposited on a virgin stigma of i et al. . For thii et al. , Fan andi et al.  and Paudi et al. ,19. To ean et al.. We usedR. alpina started from the end of May and persisted up to the end of July, with peak blooming from early June to late June. Anthesis occurred in early morning (before 8:00 AM) and soon anthers became ready to dehisce and stigma became receptive. Thus, R. alpina showed consistency for diurnal pollination. Stigma remained receptive until the complete wilting of flowers. All the floral traits of R. alpina did not differ significantly across the three study sites  was observed frequently visiting the flowers of R. alpina to feed on the pollen grains  or interspecies movement . In the year 2015, within 12 hours (from 7:00 AM to 19:00 PM), we observed a total of 154, 237 and 163 visits of beetle to the flowers of R. alpina at plot1, plot2 and plot3 respectively. Similarly, in the year 2016, within 12 hours (from 7:00 AM to 19:00 PM), we observed a total of 129, 163 and 201 visits of beetle to the flowers of R. alpina at plot1, plot2 and plot3 respectively. The visitation frequencies of beetles to the flowers of R. alpina did not differ significantly among plots, between-years and year-plot interactions , were 0.11±0.03 and 0.12±0.03 visits flower-1 hour-1, for 2015 and 2016 respectively.The visits of beetles to the flowers of . alpina . The aveMacroglossum nycteris) to the flowers of R. alpina for nectar feeding .Among the six pollination treatments, emasculated and bagged flowers did not set fruit and seed indicating the absence of apomixis. However, the rest of the treatments  set fruits and seeds, showing that R. alpina did not differ significantly between years, treatments and year-treatment interactions  is an effective pollinator of R. alpina. The percentage of fruit set and seed number per fruit in emasculated-beetle pollination, natural pollination and autonomous self-pollination did not differ significantly between years, treatments and year-treatment interactions , indicating that the pollination capacity of the beetles is independent of year factor. A total of 84.6%  and 90% (n = 40) of emasculated flowers set fruits in the year 2015 and 2016 respectively. The average number of seeds per fruit was 53.5±1.4 (n = 33) and 51.2±1.5 (n = 36) for the year 2015 and 2016 respectively. While, the emasculated flowers that were excluded from beetle’s visits did not set fruit and seed for either of the years . This reR. alpina during its single visit was 763 ± 75 and 788 ± 86 in the year 2015 and 2016 respectively. The number of pollen grains deposited by a beetle on the virgin stigma of R. alpina was several times greater than the number of ovules per flower  and were 89.57±23.65 and 94.82±23.78 for 2015 and 2016 respectively. This indicates that an individual beetle during its single visit to a flower can successfully affect pollination in R. alpina.The average number of pollen grains deposited by a beetle on a virgin stigma of r flower . PollinaR. alpina. These data indicate that the moths do not contribute to the pollination success of R. alpina though it visits the flowers for nectar feeding.The emasculated flowers visited by moths did not set fruit and seed. Similarly, the moth did not deposit any pollen grains on the stigma when it visited the flowers of R. alpina are spatially separated with stigma lying above the anther. Hence, R. alpina similar to other Himalayan Roscoea species [R. alpina. Our observations indicate that in a majority of flowers there is gradual shrinkage of style from the second day of anthesis. On the third day of flowering, the stigma is completely overtopped by the surrounding anther lobes making clear contact between anther and stigma. Our data suggest that shrinkage of style occurs at ca 2mm to allow self-pollination. Thus, R. alpina despite clear spatial separation between anther and stigma during the initial hours of flowering, undergoes autonomous selfing by the shrinkage of style and ensure the reproductive success if no pollinator vector has already provided effective pollen transfer. Our results also indicate that majority of autonomous selfing occurs on the third day of anthesis while there was no autonomous selfing on the first day of anthesis which further supports that autonomous selfing occurs by the gradual shrinkage of style on the succeeding days of anthesis. This pollination mechanism is partly consistent with the pollination mechanism in R. scheneideria which achieves autonomous selfing by the curling of hooked stigmas towards the dehisced anthers [R. debilis which assures reproductive success via autonomous selfing aided by the secretion of stigmatic fluids [R. alpina, the highest elevational ginger on earth [Like other species of the family Zingiberaceae, anther and stigma in  species  seems to anthers  and R. dc fluids . We concon earth , is one on earth .R. alpina, which lacks cantharophily syndromes. Like other members of the genus, R. alpina also exhibits strikingly long tongued fly pollination syndromes. Somewhat surprisingly however, we observed a beetle as the exclusively observed legitimate pollinator of this small alpine ginger in the Himalaya of Nepal. Our extensive observation across the study site that encompasses more than 5000 flowering individuals indicated that the only other visitor to the flowers of R. alpina were rare visits by the moth (Macroglossum nyctersis). Although, we recorded very few visits of the moth to the flowers of R. alpina, it never made legitimate contact with the reproductive parts of the flower. Moreover, our data also suggest that moth visited flowers neither received any pollen grains on their stigma nor set any fruit/seed. Thus the moth is a nectar robber of R. alpina rather than a potential pollinator. Despite the apparent absence of cantharophily syndromes, it is possible that the unelaborated and open construction of flower in R. alpina may have provided easy access to the beetle as in other beetle pollinated flowers [Beetle-pollinated flowers are typically characterized by the presence of discernible fragrance that acts as a primary attractant . Other r flowers . Althoug flowers , to the  flowers ,22,23,46Mylabris sp. is the sole observed pollinator of R. alpina in the Nepalese Himalaya, it is interesting to consider how these beetles may orientate to the flowers. Whilst beetle pollination is classically thought to be mediated by scent [Sitophilus zeamais beetle, which is classified as a pest, indicates an innate orientation in some adult beetles mediated by colour visual information [Hycleus spp. (Coleoptera: Meloidae) suggests they may damage flowers [R. alpina. Coloured trap experiments with some flower visiting beetles suggest that colour is used in a functional way that has driven the evolution of flower colouration [Pygopleurus israelitus) of that region [Pygopleurus israelitus is of family Glaphyridae of suborder polyphaga and infraorder Elateroidae for which there have been several reports of trichromacy [Trilobium castaneum superfamily of Tenebrionidae of order polyphaga most likely only possess dichromatic visual systems [Mylabris sp. in the current study belongs to family Meloidae of infraorder Cucujiformia; and also belongs to same superfamily Tanebrionidae, thus dichromatic vision would be a plausible visual system for these beetles, although the diversity of colour vision within beetle pollinators may vary spatially [Meligethes aeneus fabricius  appears to also conform to dichromatic vision potentially enabling colour choices within Tanebrionidae [Mylabris sp. may view flowers considering the likely UV (330-360nm peak range) and ‘Green’ (530-560nm) dichromatic vision [R. alpina to facilitate this. Indeed, the spectral data in Given the new finding that by scent , flowersby scent  and so vby scent ,48, and ormation . Interes flowers ,51, we douration ,52. In tt region ,52,53. Pchromacy ; however systems . The Neppatially ,48,53. Trionidae . Using trionidae  of usingc vision  within tR. alpina employs dual pollination mechanisms to help maximize its reproductive success in a sub-alpine zone of unreliable pollinator service. Our results suggest that the evolution of autonomous selfing as a predominant mode of reproduction coupled with beetle pollination system in R. alpina provide reproductive assurance to this alpine ginger. This result provides the first experimental evidence of beetle pollination system in Zingiberaceae. Indeed, the involvement of a beetle (Mylabris sp.) as the only pollinator of R. alpina suggests that a novel type of plant-pollinator interaction may be present to what has previously been considered for beetle pollination, and indicates that beetles play an important role for improving the genetic diversity of R. alpina in the Nepalese Himalayas. Flower spectra for R. alpina is different to ‘red’ signals classically associated with beetle vision, suggesting a different type of visual processing may operate in this plant-pollinator interaction at high altitudes.We show that, different to classic pollination syndromes, S1 TableMylabris sp.) to the flowers of R. alpina.Result analyzed with two way ANOVA showing the effect of years and plots on visitation frequency of a beetle ((DOCX)Click here for additional data file.S2 TableR. alpina in 2015 and 2016.Result of generalized linear model to examine the difference in fruit set percentage and seed number per fruit between autonomous self-pollination and natural pollination in the flowers of (DOCX)Click here for additional data file.S3 TableR. alpina in 2015 and 2016.Result of generalized linear model to examine the difference in fruit set percentage and seed number per fruit between emasculated-open and naturally pollinated flowers of (DOCX)Click here for additional data file.S4 TableR. alpina in 2015 and 2016.Result of generalized linear model to examine the difference in fruit set percentage and seed number per fruit between hand self-pollinated and hand cross pollinated flowers of (DOCX)Click here for additional data file.S5 TableR. alpina in 2015 and 2016.Result of generalized linear model to examine the difference in fruit set percentage and seed number per fruit among natural pollination, autonomous selfing and emasculated-beetle pollinated flowers of (DOCX)Click here for additional data file.


Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is scarce and controversial. Despite its limitations, the colour changing units (CCU) assay is still considered the golden standard titration technique for M. hyopneumoniae culture. Thus, the aims of the present study were: (1) to describe the growth dynamics and kinetics of pathogenic and non-pathogenic M. hyopneumoniae strains, and (2) to monitor the strains’ daily growth by ATP luminometry, CCU, colony forming units (CFU), and DNA quantification by real time quantitative PCR (qPCR) and by fluorescent double-stranded DNA (dsDNA) staining, to evaluate them as putative titration methodologies. The growth of the non-pathogenic J (ATCC®25934™) type strain and the pathogenic 11 (ATCC®25095™) reference strain and 232 strain was modelled by the Gompertz model. Globally, all three-strain cultures showed the same growing phases as well as similar maximal titres within a particular technique, but for CFU. However, the J strain displayed the fastest growing. During the logarithmic phase of growing, CCU, ATP and M. hyopneumoniae copy titres were strongly and linearly associated, and correlation between techniques could be reliably established. In conclusion, real-time culture titration by means of ATP or molecular assays was useful to describe the in vitro growth of the tested strains. Knowledge about the in vitro growth behaviour of a specific strain in a specific medium may provide several advantages, including information about the time required to reach maximal titres by the culture. Noteworthy, the obtained results refers to the three strains used, so extrapolation to other M. hyopneumoniae strains or culture conditions should be made cautiously.Information on the in vitro growth of pathogenic and non-pathogenic  Mycoplasma hyopneumoniae (M. hyopneumoniae) strains 11 and J, respectively /Y) × 100 [Model goodness of fit was evaluated according to the Y) × 100 . In ordeM. hyopneumoniae strain mean value of the parameters obtained from the Gompertz modelling were tested for statistical significance by Kruskal–Wallis test  with post hoc multiple comparisons (Dunn’s multiple comparison test). Normal distribution and the constant variance were evaluated by the Shapiro–Wilk’s test and Levene’s test, respectively. p values < 0.05 were considered to be significant.Statistical analyses were performed using STATISTICA version 8.0 . Differences between each M. hyopneumoniae culture titration techniques was carried out on the log2 transformed data during each strain maximal culture growth, namely logarithmic (log) phase. Thus, the period of study days compared was established taking into account the log phase duration from the growth response determined by the ATP assay in each strain culture. From such analysis, regression formulae were acquired to enable conversion of data (culture titre) obtained by means of different techniques. All statistical analyses were carried out using STATISTICA version 8.0 . The significance level (p) was set at 0.05 with statistical tendencies reported when p < 0.10.In order to compare the different assessed methodologies, a linear regression analysis between each pair of M. hyopneumoniae J strain culture at D7. Therefore, from D7 onwards, different methodology titres were assessed for J strain culture only from the replicate B. Microbial growth curves obtained for each strain and each technique were log2 transformed for the purposes of graphic representation.Negative control (uninoculated ATCC broth medium) had similar ATP values and dsDNA fluorescence reactions than the blank throughout the study (from D0 to D14). Additionally, no colonies were found in any of the agar plates inoculated, no colour change was detected at any time point and qPCR results were also negative. Unfortunately, a fungus-like contamination was detected in replicate A of M. hyopneumoniae strains is represented in Figure M. hyopneumoniae strain culture. By means of ATP luminometry, all strains showed growth curves with log, stationary and death phases, and culture colour change  firstly detected at the end of the logarithmic phase; when yellow, cultures were established in the stationary phase. M. hyopneumoniae J strain reached earlier the stationary phase than 11 and 232 strains. Nonetheless, the latter ones had longer stationary phases followed by less sharp senescence phases in comparison with J strain. Additionally, all three evaluated strains reached similar maximal raw ATP concentrations (1395.5 ± 148.9 pmol ATP/mL) once in the stationary phase.The growth response determined by ATP for each of the three M. hyopneumoniae strains is represented in Figure 9 ± 1.30 × 109 CCU/mL) in the stationary phase. Those were also reached earlier by J strain, which again showed a shorter stationary phase than M. hyopneumoniae 11 and 232 strains.The growth response determined by the CCU assay for each of the three M. hyopneumoniae J, 11 and 232 strains. Colonies from such three strains were phenotypically indistinguishable between them through microscopic visualization. Thus, end-point colonies ranged in size, from 100 to 240 µm, and were rough with irregular margin, normally lacking a clearly defined nipple (no “fried egg appearance”)  than the ones corresponding to the 11 strain culture for all the assessed techniques but for CCU. Indeed, none of the parameters obtained from the application of the Gompertz model to the CCU data was significantly different between M. hyopneumoniae strains.The Gompertz model could not be reliably applied to CFU titres because of the lack of data for 11 and 232 p values lower than 0.05 and R2 values between 0.83 and 1. In general terms, mean APE values of each strain and technique were negative, indicating overall overestimated predictions by the model. Calculated mean APE technique values  are shown in Figure M. hyopneumoniae culture titre at each time showed that differences between predicted and actual titre values alternated in positive and negative sign in all procedures. The highest error, however, was observed in the CCU method. For all the other assessed techniques.All Gompertz regression models had M. hyopneumoniae strain cultures and these were statistically associated (p < 0.001)  between all the other evaluated methodologies in all three 1) Table . EquivalM. hyopneumoniae J, 11 and 232 strains was evaluated by the application of different techniques with the potential of being used as an alternative to CCU for M. hyopneumoniae culture titration. Growth curve kinetics and dynamics of M. hyopneumoniae J strain were different from the observed for 11 and 232 strains, which, in turn, showed very similar growth curves regardless the technique applied. Thus, growth curve plots revealed a faster in vitro growth of the J strain in comparison with 11 and 232 strains as well as a straighter log phase, and a shorter stationary phase followed by a steeper senescence phase. Nonetheless, raw data analysis showed no differences between strains in terms of maximal titres reached within a particular technique.In the present study, the course of the in vitro growth of M. hyopneumoniae loses its capacity to divide (requisite for the CCU titration) before cell lysis and intracellular ATP impairment [M. hyopneumoniae growth phase in which medium turn colour may vary depending on different conditions, such as the medium itself or even the batch of medium used [Results obtained by those methods that indirectly measure the number of metabolising cells (i.e. CCU and ATP assays) indicated that, once maximal levels were reached, CCU titres dropped earlier than the onset of decrease in ATP titres. This fact supports that pairment , 27. Conium used , the volM. hyopneumoniae colonies were seen for all three strains assessed. Nevertheless, a growth curve based on CFU could only be determined by J strain, since growth of 11 and 232 colonies was much more limited. These results indicate that solid medium might support differently the growth of diverse M. hyopneumoniae strains. Indeed, M. hyopneumoniae growth on solid medium has been considered particularly difficult [M. hyopneumoniae, suggesting that agar inhibits the growth or sequesters essential nutrients for growing [M. hyopneumoniae growth curve could be accurately determined by using viable counts on the solid medium [5 CFU/mL at D6, which approximately corresponded to the medium colour shift to orange as well as with the end of the log phase determined by ATP culture concentrations. After this point, there was a decline until D10, from where no more colonies were observed. For 11 and 232 strains, colonies appeared when cultures were in the stationary phase (data not shown). Although depending probably on several conditions such as the M. hyopneumoniae strain or the medium used, overall, obtained results suggest that the CFU technique underestimates the number of live cells, suggesting the inappropriateness of this method for M. hyopneumoniae titration.In the present work, ifficult  and soli growing . In a red medium  and suchR2, the Gompertz model was well adjusted to data coming from the application of different titration techniques in all three strain cultures, except for the CFU. As a reference, former application of the Gompertz model to M. hyopneumoniae ATP growth curves resulted also in high R2 values [M. hyopneumoniae growth. In the present study, the lack of data at early phases  was perhaps a major limitation of this model. Moreover, CCU was the worst predicted method by the model, probably because progression along time did not always correspond to a further dilution turning colour, not showing a typical bacteria growth curve.Microbial growth curve is typically depicted in terms of log numbers and it has a characteristic sigmoid shape. Thus, one way to describe bacteria growth is using nonlinear regression models, such as the Gompertz equation , 30. Thi2 values . OverallM. hyopneumoniae strains is scarce and can often be difficult to interpret. In fact, the information available on such respect is up to now contradictory [M. hyopneumoniae J strain protein expression profile was found to be related to metabolism (indicative of a non-infective proliferate lifestyle that would fit a better adaptation to the in vitro conditions) whereas proteins expressed by pathogenic strains (e.g. 232 strain) were predicted to be more associated to the infectious competence [M. hyopneumoniae isolate when compared to a low virulent one [M. hyopneumoniae, although further studies are needed to confirm results and reinforce this hypothesis.Information related to the association between the in vitro growth and the difference in pathogenicity or virulence between adictory –13. In tmpetence , 11. Thimpetence , 15. In lent one . Later olent one , geneticns e.g. 2 strain wlent one  and did M. hyopneumoniae growth response than the isolate by itself [M. hyopneumoniae are hindered by the inherent difficulty in obtaining strain initial inoculum with the same amount of M. hyopneumoniae live cells. In the present case, all strain initial inoculum came from single colonies and were used when reached similar maximal ATP values. Moreover, such inocula were equally diluted at D0 and volumes taken at every time point were the same for all strain cultures. The abovementioned aspects may also partly explain the differences found in the kinetics of the growth curves between this work and a previous one [M. hyopneumoniae in vitro growth. Besides dissimilarities, it is important to note that previous estimated maximum ATP titres reached values of 1350 pmol ATP/mL [Although reasons for the former inconsistencies are difficult to elucidate, some aspects should be kept in mind. First, the growth medium has been reported to have a more pronounced effect on the y itself . Second,ious one . While tM. hyopneumoniae strains as well as culturing time points should be included to better assess the relationship between techniques. Nevertheless, growth kinetics of 7 diverse M. hyopneumoniae isolates were previously judged and, in agreement with the present results, CCU and ATP titres were strongly linearly linked during log phase [M. hyopneumoniae culture had a mean ATP content of 4.17 × 10−3 pmol per colour change of 1 mL culture, whereas another study reported M. hyopneumoniae culture to contain 1.77 × 10−6 pmol ATP per CCU in 1 mL of culture [M. hyopneumoniae, which might be problematic when other inoculum type rather than culture is used (e.g. lung homogenate). In this case, M. hyopneumoniae copies per mL assessed by qPCR may be the most approximated value to the number of M. hyopneumoniae cells. The amount of M. hyopneumoniae DNA determined by qPCR revealed that 1 CCU/mL correspond to 1260 mycoplasmas. This value is close to previous data reporting that 1 CCU/mL of M. hyopneumoniae strain 116 corresponded to 1000 mycoplasmas [M. hyopneumoniae log growing phase, its inability to differentiate between alive and dead cells is a major disadvantage during the later stages of the growth. Given that each of the evaluated titration methodologies have limitations, their use in combination is probably required to optimise M. hyopneumoniae culture titration accuracy.Comparison of results obtained by the different applied techniques was an important part of the present study. During the log growth phase, titration results of the four assays were highly and significantly correlated and a strong linear relationship was observed. Ideally, further og phase . Accordi culture . Hencefooplasmas . While qM. hyopneumoniae growth protocols in the laboratory as well as inoculum production for experimental applications, it is important to know the in vitro growth kinetics and dynamics of a particular strain in a particular medium. Once this information is known, a more reliable approximation of the growing phase status from the culture colour change can be performed. For this purpose, ATP is an economical, rapid, timely and efficient assay that allows the distinction of the different M. hyopneumoniae growing phases in culture. Nonetheless, ATP is not a M. hyopneumoniae specific technique; thus, its combination with a specific detection method (such the qPCR) might be useful. Lastly, further studies with more strains, both at in vitro and in vivo levels, are needed to elucidate the putative relationship between M. hyopneumoniae in vitro behaviour and virulence.In order to standardize