Oligonychus afrasiaticus (McGregor) is an important pest causing substantial economic losses to date palm fruits (dates). The application of mycopathogens with plant secondary metabolites, which may proceed synergistically is thus essential to augment sustainable management strategy for O. afrasiaticus. In this regard, extensive laboratory experimentation involving compatibility, synergism, and host defense was performed to develop stable pest management option. The toxin-pathogen compatibility assay results revealed compatible interaction  of B. bassiana ARSEF 8465 against each tested concentration of commercially available (+)-α-Pinene that provide the opportunity to further explore the time and concentration dependent mortality and defense related enzymatic regulation analysis. The time-mortality response assays that mainly comprised of various proportions of B. bassiana ARSEF 8465 and (+)-α-Pinene revealed that the sole application of B. bassiana ARSEF 8465 (LC50 = 19.16 mg/mL), and (+)-α-Pinene (3.41 mg/mL) found to be least lethal compared with joint applications (LC50 ranged from 1.32–7.06 mg/mL). The treatments complied under Scheme IV (80% (+)-α-Pinene: 20% B. bassiana ARSEF 8465 Conidia) led to strong synergistic interaction (joint toxicity = 755). In addition, synergistic interactions greatly induced enzymatic activities of the studied antioxidants (CAT and SOD), and defense-related enzymes (GST and AchE). We concluded that join application of B. bassiana ARSEF 8465 and (+)-α-Pinene is a promising option for controlling Oligonychus afrasiaticus populations. Phoenix dactylifera L.) is an economically important palm species grown in the arid, sub-tropical, and tropical regions. It has the tendency to tolerate not only the harsh extremely high desert temperature but also resistant to drought and salinity. Globally, Saudi Arabia stands second for dates production after Egypt. Their production in Saudi Arabia according to United Nations Food and Agriculture statistics reached 1.54 million tons/year during 2019 that is 16.97% of the total world’s dates production [Oligonychus afrasiaticus (Acari: Tetranychidae), is an economically important pest of the fruit of date palms in Saudi Arabia [O. afrasiaticus on date palm fruits [O. afrasiaticus infestations start on the date palms by spinning the web around the bunches of dates [The date palm  evaluating the compatibility of conidial suspensions with (+)-α-Pinene by compatibility assays; (2) testing the acaricidal potential of a selected plant secondary metabolite (+)-α-Pinene, and B. bassiana ARSEF 8465 by concentration mortality response bioassays; (3) calculating the joint toxicity index to sort out the nature of their interaction; (4) exploring the treatment effects on the regulation of defense-related enzymes controlling physiological mechanisms of date palm dust mites, in order to develop a synergistic interaction with improved efficacy for the effective management of O. afrasiaticus populations infesting date palm fruits.In the past, little information is available on the joint action of plant secondary metabolites with conidial suspension against siaticus ,37. ThesBeauveria bassiana ARSEF 8465 originally isolated from Doru lineare during December 2006 from Argentina was procured from the ARSEF collections of USDA-ARS. The Beauveria bassiana ARSEF 8465 isolate selection for this particular study was mainly on the basis of preliminary laboratory bioassays that showed virulence against O. afrasiaticus. The cultures of B. bassiana ARSEF 8465 isolate were maintained on Petri plates provided with potato dextrose agar in complete darkness for 24 d at 25 ± 0.5 °C. For bioassays, conidial suspensions were prepared in Tween 80 at a strength of 0.05% by Neubauer hemocytometer under a compound microscope.The culture of The field collected date palm dust mite populations from National Center for Palms and Dates, Al-Ahsa, Saudi Arabia were maintained until experimentation in a photoperiod of 16 h light and 8 h darkness . Overall, five different populations of date palm dust mites were directly collected from the mentioned locality to prepare different replicates for this study.The compound (+)-α-Pinene as plant secondary metabolite was purchased from Sigma Aldrich London, UK (Cat # 268070) for toxicity, compatibility, toxin-pathogen interactions, and host physiological defense related enzymatic analysis. The stock solution of (+)-α-Pinene  was prep7 conidia/mL) of B. bassiana ARSEF 8465 was evaluated by calculating the biological index (BI) through below mentioned formula [The compatibility of plant secondary metabolite (+)-α-Pinene with the conidial suspension -α-Pinene, separately. Ten Petri dishes (n = 10 replicates) each by pouring 10 mL of potato dextrose agar supplemented with each tested concentration of (+)-α-Pinene was prepared under a laminar airflow cabinet. The control treatment petri dishes were prepared using PDA with 1% ethyl alcohol. After cooling the petri dishes, 200 µL conidial suspension of B. bassiana ARSEF 8465 (1 × 107 conidia/mL) was used to inoculate all experimental units. After sealing with parafilm, all the inoculated petri dishes were incubated (25 ± 0.5 °C) in complete darkness. After 12 h post-inoculation, percent conidial germination was recorded under compound microscope after counting 100 conidia from ten different fields of vision. Germination data were adjusted through arcsine transformation prior to one way analysis of variance (ANOVA) to analyze percent conidial germination (GR), and significant differences among them by applying Fisher’s LSD test [In the first part of compatibility assays, impact of various concentrations  of (+)-α-Pinene on the LSD test .B. bassiana ARSEF 8465 as described in our previous study [n = 10 replicates) were poured with PDA supplemented with each concentration of (+)-α-Pinene. The control treatment was prepared by using PDA with 1% ethyl alcohol. Each experimental unit petri plate was inoculated by micropipette with five microliter conidial suspension of B. bassiana ARSEF 8465 (1 × 107 conidia/mL). Two weeks post-inoculation at 25 ± 0.5 °C, vegetative growth (VG) data of the fungal growth were measured with the help of a transparent ruler to calculate their perpendicular radial lengths. One way Analysis of variance (ANOVA) was used to analyze vegetative growth of B. bassiana ARSEF 8465, and significant differences by Fisher’s LSD test [In the second part of compatibility assays, effect of (+)-α-Pinene at different concentrations was evaluated against the vegetative growth of LSD test .B. bassiana ARSEF 8465 exposed with above mentioned concentrations of (+)-α-Pinene was determined from 15 mm colony suspended in Tween 80 at a strength of 0.05%. The conidiation was determined from above mentioned two weeks old fungal cultures used to record vegetative growth. The conidial count of each experimental unit suspension was made by a Neubauer hemocytometer under a compound microscope. The conidiation data were analyzed by one way ANOVA, and significant differences by Fisher’s LSD test [In the third part of compatibility assays, conidiation of LSD test .B. bassiana ARSEF 8465, and their different combinations indicated in O. afrasiaticus deutronymphs stage [O. afrasiaticus were transferred by camel hair brush separately on each treated leaf-disk. Five replicates per treatment were prepared likewise by incubating each experimental unit at 62.5 ± 12.5% RH; 25 ± 1 °C. Similarly, control treatments were also prepared likewise using their respective solvents. Mortality data for each experimental unit was recorded after every 24 h under the microscope. Abbott formulae was used to correct the percent mortality data of O. afrasiaticus [50 determination by Probit analysis [The impacts of the sole application of (+)-α-Pinene and hs stage . Differesiaticus . The coranalysis , for subanalysis . Howeveranalysis .O. afrasiaticus. The exposed individuals of the five replicates including control treatments were incubated until 96 h post-exposure on controlled conditions. Approximately 500 alive date palm dust mites after 96 h were removed to prepare their solution in the pH 7.0 potassium phosphate buffer (0.05M). After centrifugation, each treatment supernatant was used to quantify protein at 595 nm absorbance [The date palm leaf-disks as mentioned in sorbance . The enzsorbance . Each trsorbance , and recsorbance .B. bassiana ARSEF 8465, and revealed directly proportion concentration-dependent response  in percent germination of B. bassiana ARSEF 8465 conidia recorded 12 h post-inoculation in the presence of all tested concentrations of (+)-α-Pinene. Overall, we have calculated negligible germination inhibition response that ranged from 0.91% for the lowest concentration (0.7 mg/mL), to 3.74% for the highest concentration of (+)-α-Pinene (3.5 mg/mL), compared to the control treatment.All the tested concentrations of (+)-α-Pinene affected the studied parameters of response . There wB. bassiana ARSEF 8465 in response to different concentrations of (+)-α-Pinene such as 0.7, 1.4, 2.1, 2.8, 3.5 mg/mL resulting 1.36%, 4.04%, 7.16%, 11.32%, and 14.32% inhibition compared to control treatment, respectively. However, mean vegetative growth of B. bassiana ARSEF 8465 recorded against each tested concentration showed highly significant differences  as shown in Similarly, vegetative growth also revealed similar inhibition patterns of B. bassiana ARSEF 8465 also showed similar pattern in response to different concentrations of (+)-α-Pinene. Results exhibited significant differences among treatments . Furthermore, we have recorded 5.26%, 11.84%, 15.79%, 23.68%, 30.26% conidiation inhibited against various concentrations of (+)-α-Pinene such as 0.7, 1.4, 2.1, 2.8, and 3.5 mg/mL, respectively.Conidiation of B. bassiana ARSEF 8465 response at all tested concentrations of (+)-α-Pinene revealed compatible interaction . However, mortality of O. afrasiaticus showed significant differences in response to conidial suspensions of B. bassiana ARSEF 8465 , concentrations , and their interaction . Similarly, sole application of (+)-α-Pinene also exhibited concentration-dependent mortality response , recorded mortality at different time intervals , and their interaction  revealed significant differences. The LC50 value was found to be 3.41 mg/mL.The current study revealed that all the treatments whether a sole application or their combined application were lethal to the st mites . The B. SEF 8465  recordedresponse . The expB. bassiana ARSEF 8465 with different concentrations of (+)-α-Pinene evaluated by four different Schemes revealed combined treatment enhanced mortality effect in all the studied interactions (B. bassiana ARSEF 8465 Conidia) bioassay results exhibited Antagonistic interaction by revealing a joint toxicity index score of 58 , among all the interaction treatments , with different concentrations , and their interaction , exhibited significant differences  different concentrations , recorded at different time intervals , and their interaction . The Scheme II bioassay results revealed synergistic interaction (joint toxicity index = 121) as shown in There was a significant difference in the cumulative percent mortality  of O. afO. afrasiaticus treated with Scheme III bioassays treatments exhibited synergistic interaction by revealing high (280) joint toxicity index . However, different concentrations , resulted in the significant differences in the mortality of O. afrasiaticus recorded at various time intervals . Furthermore, interaction , also revealed significant differences , compared with other interactions , against different concentrations , and their interaction .The simultaneous exposure of (+)-α-Pinene and siaticus . The treractions . The anaO. afrasiaticus measured 96 h post-exposure exhibited significant differences . Overall, the sole application of conidial suspension of B. bassiana ARSEF 8465 at different concentrations failed to induce very high CAT activities, and resulted in less than 10% CAT activities relative to control treatment , and their interaction .The catalase (CAT) activities of reatment . HoweverO. afrasiaticus exposed with various concentrations , treatments, , and their interaction  exhibited significant differences. The most effective treatment (80% (+)-α-Pinene: 20% Conidia) determined in the current study on the basis of lowest LC50 value (1.32 mg/mL) greatly induced the activities of SOD and kept at a higher level of significance in comparison with rest of the treatments (O. afrasiaticus fed with sole application of (+)-α-Pinene, and the treatment with 20% (+)-α-Pinene: 80% Conidia, remained at a lower level of significance in comparison with the rest of the treatments.The relative activities of Superoxide dismutase (SOD) measured among eatments . On the O. afrasiaticus with various treatments , compiled under different concentrations , and their interaction , exhibited highly significant differences , complied with different concentrations , and their interaction  showed highly significant differences (Relative activities of glutathione S-transferase (GST) from ferences . The treferences . The relferences .B. bassiana ARSEF 8465, and their dual application found to be an attractive alternative approach to circumvent slow mortality criticism by producing strong synergistic interaction, which tremendously induced the target host defense-related enzymatic regulation, and ultimately increase the mortality of O. afrasiaticus.The development of effective and stable pest management option involves the extensive toxin-pathogen compatibility and target host time- and concentration-dependent mortality response experimentation. This study clearly showed that the addition of (+)-α-Pinene found to be compatible with the B. bassiana ARSEF 8465 evaluated here against date palm dust mites exhibited the highest LC50 value (LC50 = 19.16 mg/mL). Furthermore, none of the tested conidial concentration able to impart 100% mortality, and their recorded cumulative mortality even at the highest concentration found to be <75%. The slow mortality response revealed here in the current study by the fungal conidial suspensions is in agreement with previous studies aiming to screen the most virulent isolate against various pest species [Naturally-occurring entomopathogenic fungi are well-known to infect the populations of pests ,45,46,47 species ,45. Thes species , or by iSitophilus zeamais Motschulsky [Spodoptera litura and Achaea janata [O. afrasiaticus exhibited enhanced mortality response over time and concentrations in the current study. These results coincide with previous research findings [S. zeamais Motschulsky. However, the low toxicity response (LC50 = 3.41 mg/mL) recorded in this study is in compliance with previous studies against date palm dust mites exposed with some other plant secondary metabolites including 1-chlorooctadecane [B. bassiana ARSEF 8465 against all tested concentration was high, ranging from 95.00 to 98.20% , and 74.20 to 85.40 mm (vegetative growth). The biological index score (BI ranged from 79–95) of B. bassiana ARSEF 8465 calculated against each tested concentration of (+)-α-Pinene demonstrated that their joint application is suitable to develop synergistic interaction against O. afrasiaticus populations.Plant secondary metabolites are untapped chemicals reservoir currently emerged as an attractive management option for agricultural pests due to the advancement in analytical chemistry and least risk to the environment. The studied (+)-α-Pinene is a polyphenolic terpene hydrocarbon with known toxicity against various pests including head lice , Sitophischulsky , Spodopta janata . The confindings  that strtadecane , and phytadecane , and theO. afrasiaticus populations control in the date palm plantations, the importance of compatible toxin-pathogen interaction is highly applicable to facilitate the development of eco-friendly products. Our results revealed that pairing of B. bassiana ARSEF 8465 with (+)-α-Pinene in different proportions exhibited different responses among exposed date palm dust mites. However, the bioassay schemes with a higher proportion of (+)-α-Pinene contribute to enhanced mortality response, which ultimately lead to synergistic interaction. The enhanced mortality response among the treatments with a higher proportion of compounds recorded in the current study coincides with previous studies reporting the incorporation of phytol within the conidial suspensions tremendously enhance the combined treatment effect by revealing a very high score of joint toxicity index (691) [B. bassiana and commercial biopesticide against Colorado potato beetle larvae further strengthened the current findings [B. bassiana ARSEF 8465 Conidia) depicted strong synergistic interaction (joint toxicity index = 755). These results are further supported by a previous study [For successful ex (691) . The enhfindings , and allus study , which sB. bassiana ARSEF 8465 Conidia) showed enhanced mortality response compared with O. afrasiaticus exposed to (+)-α-Pinene or B. bassiana ARSEF 8465 sole application, but without synergistic interaction. However, both the tested fractions interacted antagonistically (joint toxicity index = 58) that might because of low proportion of (+)-α-Pinene. The current interaction data agree with Al-mazra’awi at al. [B. bassiana and neem tree extracts although showed high mortality, but revealed antagonistic interaction. Taken together, these findings suggested that careful synthesis of joint application is critically important to shift antagonistic interaction towards strong synergistic interaction to develop strong toxin pathogen synergy for effective and efficient management of agricultural pests.The joint application of conidial suspension and chemical compounds, which mainly aimed at improving the treatment effect might also lead to antagonistic interaction that limits the combination of the applied mixture . In our i at al. , who shoAntioxidant defense system is important to scavenge the reactive oxygen species (ROS) generated within the target host as a result of the exposure to toxin/pathogen. The enzymes including SOD and CAT that prevent the accumulation of ROS explored in the current study depicted significantly high activities compared to control treatment after 96 h post-exposure. The increase in CAT and SOD activities is consistent with previous studies ,43, reveB. bassiana ARSEF 8465 conidia against date palm dust mites are consistent with those of Zibaee and Bandani [Eurygaster integriceps Puton exposed with Artemisia annua. These findings allowed us to suggest that the enzymes involved in defense mechanism play a pivotal role as these mechanisms utilize most of the host energy to defend the toxin or pathogen. The host could withstand these circumstances to certain limits and these conditions ultimately led the target host towards death.Host defense mechanisms in the context of toxin-pathogen interaction studies are very much pertinent because these interactions act as drivers to impart physiological stress among target hosts. Obtained results indicated that synergistic effect greatly induced the relative GST and AchE activities compared to control treatment. The enhanced defense-related enzymatic response revealed especially from the interaction treatments (synergistic interactions) with a higher proportion of % (+)-α-Pinene represents date palm dust mites response, which ultimately leads to strong physiological stress also confirmed by previous studies conducted on the host defense mechanism ,55,56,57 Bandani , who repB. bassiana ARSEF 8465 that greatly induced the activities of antioxidant (CAT and SOD), and defense-related (GST and AchE) enzymes, and ultimately imparted higher mortality of date palm dust mites. Therefore, the joint application of (+)-α-Pinene and B. bassiana ARSEF 8465 conidial suspensions based bio-control agents could provide a greater opportunity for environmentally friendly practical integrated pest management strategy for Oligonychus afrasiaticus (McGregor), an important economic pest of date palm fruit (dates).In conclusion, the results of the current study demonstrated a compatible interaction between (+)-α-Pinene with


Adolescents frequently engage in noisy leisure activities which can result in hearing-related problems. However, the effect of leisure noise exposure is liable to an individual’s risk-taking behavior. Identifying leisure noise activities and relevant factors related to risk-taking behavior in adolescents, are important to optimize hearing conservation programs targeting youngsters. The purpose of the study was to explore the presence of hearing-related symptoms, as well as noise exposure during various activities, and the use of hearing protector devices (HPDs) in adolescents in two educational programs in Flanders. In addition, their attitudes and beliefs towards noise, hearing loss, and HPDs were investigated. The final sample consisted of 247 adolescents. The most important hearing-related symptoms after noise exposure were tinnitus and noise sensitivity. With regard to leisure noise exposure, listening to PMPs was most frequently reported. The use of HPDs during most noisy activities was limited, in accordance with the presence of hearing-related symptoms, considering noise as unproblematic, and having worse scores on subscales of the beliefs about hearing protection and hearing loss scale. In the future, hearing conservation programs should target adolescents specifically for a more health-orientated behavior towards noise exposure, hearing loss, and HPDs. In 2015, the World Health Organization estimated that 1.1 billion young people worldwide could be at risk of developing hearing loss due to unsafe listening practices , while nChildren and adolescents frequently engage in noisy leisure activities, such as using personal music players (PMPs) through headphones or earbuds including mp3-players, mobile phones, tablets, etc., attending discotheques and concerts, or playing musical instruments, alone or in a band or orchestra ,11,12,13In young adults, no significant differences in their hearing were found between subjects with different lifetime equivalent noise exposures based on self-report ,16,17. HIn addition, the effects of leisure noise exposure on the auditory system are liable to an individual’s risk-taking behavior. According to Widen, this behavior is based on aspects from the Theory of Planned Behavior  and HealIn young adults, it was found that their attitudes and beliefs regarding noise, hearing loss, and hearing protector devices (HPDs), had a significant impact on hearing , as wellThe aim of the study was to explore the presence of hearing-related symptoms, as well as attendance and estimation of loudness during various leisure noise activities, and the use of HPDs in adolescents in two different educational programs in Flanders. In addition, their attitudes and beliefs towards noise, hearing loss, and HPDs were investigated. We hypothesized that tinnitus and noise sensitivity after noise exposure would be reported frequently, that adolescents would participate in various leisure noise activities without using HPDs, and that their attitudes and beliefs towards noise, hearing loss, and HPDs would be positive. We also hypothesized differences in the presence of hearing-related symptoms, and beliefs towards noise, hearing loss, and HPDs between adolescents from two educational programs. This study was a cross-sectional study using a self-administered questionnaire in a group of high school students from the third grade of secondary education. The principals of four chosen high schools were contacted and asked to participate in the study. These high schools were situated in East-Flanders, which is a province in Flanders.The final sample consisted of 247 high school students , with an age range of 15 to 20 years . Furthermore, 105  and 142  students participated in the GSP and TSP, respectively. The study was approved by the Ethical Committee of Ghent University Hospital and was conducted in accordance with the ethical standards stipulated in the Helsinki declaration for research involving human subjects (BC-07045 and 29 April 2019).As the evaluation of noise exposure in young people should contain different aspects such as the evaluation of the amount of noise exposure, hearing loss, and hearing-related symptoms, as well as attitudes towards noise exposure, hearing loss, and use of HPDs, a Dutch questionnaire encompassing these factors, was needed. However, a questionnaire addressing all of these aspects through one questionnaire was lacking. Hence, Keppler et al.  designedThe final questionnaire had four sections. The first section included several socio-demographic variables such as age and gender. The second section consisted of questions pertaining to the medical history of ear-related disorders, the subjective hearing status, problems with speech understanding, and the presence of hearing-related symptoms . In the case of tinnitus, the questions were formulated as such that it would be possible to distinguish between temporary and chronic tinnitus, and that subjects indicating to have chronic tinnitus are subjects with continuous or intermittent tinnitus. In the third section of the questionnaire, participant’s participation in noisy activities, as well as their estimation of loudness in terms of communicative effort, was administered for several leisure activities that are common among young people such as visiting nightclubs, using PMPs, and playing musical instruments. Five levels of loudness were considered: (1) level of a normal conversation, (2) level of a loud conversation, (3) level at which one must shout over one meter in order to be heard , (4) level at which one must shout over a near distance in order to be heard , (5) level that makes communication impossible . FurtherThe fourth section consisted of a Dutch modified version of the ‘Youth Attitude to Noise Scale’ (YANS) ,35 and aTo ensure that the questionnaire was completed correctly by the subjects, instructions were provided at the beginning of the form as well as for each new section. All terminology regarding leisure noise, hearing, and tinnitus, as well as HPDs, was explained and appropriate examples were given.Statistical analysis was performed using SPSS version 25 . Descriptive parameters and normality analyses were established for the different questionnaire outcomes. t-test was conducted. To examine possible correlations between categorical variables, chi-square tests (2 × 2 or 2 × 3 tables) were performed. When the chi-squared test was significant, pairwise comparisons were performed for the 2 × 3 tables, using Bonferroni corrections of the p-values (α = 0.05/3). If one or more cells had an expected count of less than five, Fisher’s exact test was used. Except after Bonferroni corrections, p-values less than 0.05 were used indicating statistical significance.Subsequently, simple analyses were performed to evaluate the relation of different variables with the educational program . In the case of continuous variables, an independent samples 2 = 10.427; p < 0.05. In the majority of all students (97.5%), tinnitus was temporary and disappeared within 72 h, whereas 2.5% of the students reported chronic tinnitus. Although the difference in the occurrence of temporary and chronic tinnitus between the students from the GSP and TSP was not statistically significant , it should be noted that the students reporting chronic tinnitus were all from the TSP group. For the remaining hearing-related symptoms, no significant differences were found between the students from the GSP and TSP . p > 0.05).In addition, difficulties with speech understanding were questioned among the students. Overall, the majority of the students reported difficulties with speech understanding ‘sometimes’ for each of the different listening situations . Besidesp > 0.05) as well as their estimated subjective loudness of the activities . Although not statistically significant, descriptive data suggests that students from the TSP reported more participation in occupational noise activities and the use of noisy tools compared to the students from the GSP.The students included in this study participated in a variety of activities. The highest attendance was reported for listening to PMPs through headphones or earbuds (98.4%), watching movies or plays (97.5%), listening to music through loudspeakers (89.1%), and visiting or working at nightclubs or music venues (82.3%). Furthermore, visiting and working at nightclubs and music venues were described as the loudest, where one must shout over a near distance. Participation and self-estimated median loudness for the different activities were compared between the students from the GSP and TSP . For eacp > 0.05) (p > 0.05). Furthermore, of subjects who indicated wearing HPDs in one or more activities, the majority indicated concerns about their hearing as the main reason to wear HPDs (47.6%). Besides, 30.7% of the subjects indicated that loud music is the main reason to wear HPDs, while 4.8% wear HPDs because of a detected hearing loss. The remaining subjects (16.9%) indicated no clear reason for wearing HPDs. Reasons for wearing HPDs were also not significantly different between the students from the GSP and TSP .In addition, the subjects were asked about their use of HPDs during the activities they participate in. For all of the activities, the majority of students did not wear HPDs. The highest grades of wearing HPDs were reported for using noisy tools , attending or working at musical concerts , and visiting or working at nightclubs of music venues . No significant difference in the use of HPDs during the different activities was found between the students from the GSP and TSP  . Regardip > 0.05). In contrast, experiencing hearing loss and compulsory wearing of HPDs were the main reasons to be willing to wear HPDs, whereby no differences were found between the students from the GSP and TSP .Finally, all subjects were asked whether they were already recommended to use HPDs as well as about the disadvantages and their willingness of wearing HPDs . The majp > 0.05). In contrast, a significant difference between the GSP and TSP group was found for the scores on the factors related to the concertation in noisy environments (t(240) = −2.756; p < 0.01) as well as the intent to influence sound environment (t(240) = −2.789; p < 0.01). Specifically, students from the TSP had significantly higher scores on both these factors. t-test, p > 0.05), except for the factor related to the severity of the consequences of hearing loss (t(234) = −2.893; p < 0.01). Specifically, students from the TSP showed significantly higher scores for this factor, which indicates that students from the TSP were less aware of the severity of hearing loss compared to the students from the GSP.For the subscales of the BAHPHL, the highest and lowest average scores were respectively found for the social norms and the severity of consequences of hearing loss. No significant differences in the scores were found between the students from the GSP and TSP (independent samples The present study explored hearing-related symptoms, leisure noise exposure, and attitudes and beliefs towards noise, hearing loss, and HPDs in 247 adolescents between 15 and 20 years in two educational programs in Flanders using a self-report questionnaire. With regard to hearing-related symptoms after noise exposure, the presence of temporary and chronic tinnitus in the current study was 63.2% and 1.6%, respectively. Generally, this is lower as compared to previous studies ,40,41,42The most frequent leisure noise activities reported by the adolescents were listening to PMPs through headphones or earbuds, watching movies or plays, listening to music through loudspeakers, and visiting or working at nightclubs or music venues. As compared to young adults, the use of PMPs is higher in adolescents, consistent with previous studies ,44,45,46Regarding risk-taking behavior, attitudes and beliefs towards noise exposure, hearing loss, and HPDs were questioned using the YANS and BAHPHL in the current study. These scales evaluate the factors from the Theory of Planned Behavior  and HealIn addition to questioning attitudes and beliefs towards noise exposure, hearing loss, and HPDs in relation to confounding variables, these aspects were previously also related to hearing status. More specifically, young adults with more problematic attitudes regarding noise exposure or positively evaluating barriers to preventive action, have already significant hearing damage in comparison to those with more negative or neutral attitudes and beliefs . FinallyThe reported use of HPDs during most noisy exposure activities was limited in the current study. However, during some activities, i.e., using noisy tools, attending or working at musical concerts, festivals, nightclubs, or music venues, adolescents reported wearing HPD more as compared to previous studies in young adults  and adolEX of 80 dBA [One of the strategies to prevent damage to the auditory system by leisure noise exposure in youngsters is controlling noise levels. In Flanders, there is noise legislation for indoor and outdoor music venues, although the compliance should be increased . Howeverf 80 dBA . In addif 80 dBA ,34. Durif 80 dBA , should The results of the current study should be considered taking into account some limitations. First, the sample might not be representative of all Flemish adolescents, with regard to geographic distribution and educational programs. Nevertheless, it was ensured that two educational programs within a region in Flanders were represented. Second, a self-report questionnaire was used, in which noise exposure was estimated. Although the questionnaire considers different leisure activities in detail, it is possible that the time spent on leisure activities and the estimation of loudness are imprecise. Other measures, not restricted to prespecified activities during specific periods in life, such as the Noise Exposure Structured Interview , might bThe present study of 247 adolescents between 15 and 20 years in two educational programs in Flanders showed the presence of hearing-related symptoms such as temporary and chronic tinnitus and noise sensitivity after noise exposure. With regard to leisure noise exposure, in this population, listening to PMPs was more frequently reported, as compared to attending nightclubs or music venues. The use of HPDs during most noise exposure activities was limited, in accordance with the presence of hearing-related symptoms, considering noise as unproblematic, and having worse scores on subscales of the BAHPHL related to barriers to preventive action, behavioral intentions, and social norms. Between the adolescents in the two educational programs, differences in the presence of tinnitus after noise exposure, and subscales of the YANS and BAHPHL regarding concentration in noisy environments, the intention to influence the sound environment, and severity of the consequences of hearing loss were found. In the future, hearing conservation programs should target adolescents specifically for a more health-orientated behavior towards noise exposure, hearing loss and HPDs.


Ceruminous glands are located in the skin of the cartilaginous portion of the external auditory canal, and ceruminous gland adenoma originating from the middle ear mucosa is extremely rare. We report a case of middle ear ceruminous gland adenoma which caused long-standing otomastoiditis and mixed hearing loss with a large air-bone gap by obstructing the bony Eustachian tube. We discuss the clinical characteristics and histologic features of the present case. Cerumen, which plays an important role in protecting the ear from infection and mechanical damage, is produced by the ceruminous gland and sebaceous gland. The human ceruminous glands are modified apocrine glands and located in the skin of the cartilaginous portion of the external auditory canal , 2. The A previously healthy 56-year-old woman complained of left-side hearing loss over a 10-month period. She had been receiving treatment at another hospital and undergone ventilation tube insertion surgery in the left ear 4 month prior. The patient reported that the left-side hearing loss was not relieved after the surgery. On the otoendoscopic examination, a pinkish mass was seen through the anterosuperior quadrant of the tympanic membrane without discharge . A nonenAlthough ceruminous gland neoplasms are relatively common in other mammals , they arProgressive aural fullness, hearing loss, otalgia, and otorrhea are known as typical symptoms of middle ear neoplasm –10. Our The present study demonstrated that ceruminous gland adenoma, which originated from the middle ear, can obstruct the bony portion of the Eustachian tube and cause progressive conductive hearing loss and otomastoiditis. Thus, though extremely rare, the middle ear ceruminous gland adenoma should be taken into consideration in the differential diagnosis when conductive hearing loss and otitis media are progressively worsening and do not respond to long-term conventional treatment.


Staphylococcus aureus (MRSA) infections and enterococcal infections, which are resistant to beta-lactams and other antibiotics. Rising GPA resistance has led to the discovery and clinical development of synthetic second-generation glycopeptides but more needs to be done to stand up against the evolution of resistance mechanisms. In a recent publication in Nature Communications, a team of scientists led by Gerard Wright at McMaster University in Canada, developed a clever synthetic biology platform for the production and discovery of novel GPAs.Antibiotic resistance is one of the greatest challenges facing public health.GPAs are heptapeptides that are naturally synthesized in actinobacteria, orchestrated by co-localized genes in the genome, called biosynthetic gene clusters (BGCs), and a host of other genes for modification, regulation and transport. Although next-generation sequencing technologies and machine-learning tools have enabled the identification of a wealth of BGCs in bacterial genomes, low or no yields of GPAs from parental actinomycetes, lack of heterologous hosts for expressing BGCs and difficulty in cloning these large constructs (>70 kb) has kept these GPAs elusive.et al. engineered Streptomyces coelicolor, a genetically tractable microorganism, that contains the biosynthetic machinery and precursors required for GPA biosynthesis, including pathways for nonproteinogenic amino acid components to create the GPAHex chassis. To clone the large BGCs and increase selection efficiency, they developed an optimized transformation associated recombination (TAR) system that allows isolation and manipulation of large DNA constructs.Xu ura3 counter-selection marker, which was also optimized by introducing additional transcriptional initiation sites between the TATA box and the start codon to maximize ura3 transcription.The TAR system relies on a copy number control replicon that can be conditionally induced for high copy numbers. The system utilizes a et al. cloned a 76 kb region, constituting the peptide scaffold and six TISs, into the S. coelicolor chassis strain via E. coli-yeast triparental mating. Growth inhibition was observed against Bacillus subtilis and the production titer was 65.4 mg/l, 19-fold higher compared to the parental Streptomyces strain.To demonstrate the synthesis of a GPA, they targeted corbomycin, a Type V GPA. Corbomycin was discovered by phylogeny guided genome mining, but further development was hindered by low titers in the producer Streptomyces strain. Xu B. subtilis. Chromatographic analysis revealed that the structure of this novel GPA was indeed analogous to teicoplanin, with additional modification of specific sites. They took this discovery platform a step further by identifying a cryptic Type V GPA produced by a number of Streptomyces strains and successfully expressing the GPA with good yields. The structure revealed an aliphatic valine residue which is uncommon in other Type V GPAs, composed entirely of aromatic residues.The authors also used the GPAHex platform for the discovery of a GPA in an Amycolatopsis strain that shares homology with the teicoplanin class of GPAs but is transcriptionally inactive in the parental strain. They cloned the scaffold including four TISs into the chassis strain to express GP1416 that displayed antibacterial activity against This GPAHex system expands the repertoire of GPAs and provides a platform for the discovery and development of GPAs and other peptide natural products, especially from organisms that lack genetic tractability. The application of their optimized TAR cloning system is broad-reaching in that can be used for assembly of large constructs with higher efficiencies in many synthetic biology applications and functional/structural genomics studies.Conflict of interest statement. None declared.


Cervical cancer, one of the most common cancers affecting females in South Africa, commonly requires a cisplatin-based-treatment regimen, which has been associated with ototoxic side effects. However, cisplatin-associated ototoxicity is largely under-reported in South Africa, despite its impact of hearing loss having serious overt ramifications on the quality of life of these patients. Hence, a prospective cohort study was undertaken to assess the audiological changes in female cervical cancer patients receiving cisplatin therapy.To present details of the feasibility study and initial results on hearing patterns in cervical cancer patients receiving cisplatin chemotherapy. .Fifty cervical cancer patients commencing with cisplatin chemotherapy underwent audiological assessments at a hospital in South Africa at various time intervals. Assessments included case history, otoscopic examination, immittance audiometry, pure tone audiometry (including high-frequency audiometry), speech audiometry, and distortion product otoacoustic emission testing. Data analysis involved the use of descriptive statistics and the Cochran-Armitage trend test for a linear trend in proportions.Fifty participants, aged between 32 and 79 years , were recruited. Clinical findings revealed an incidence of 100% ototoxic hearing loss at the one-month post-treatment, i.e., 98%  after three cycles of cisplatin and 2%  at one-month post-chemotherapy. Sensorineural hearing loss and high-frequency tinnitus were most common. Deterioration in hearing thresholds was more evident in the extended high-frequency range, with the number of “no-responses,” from 11,200 Hz to 20,000 Hz, increasing with each successive audiological evaluation. This study further indicated that recruitment and follow-up of study participants within a limited resource setting are possible. However, cognizance must be given to a multidisciplinary approach and constant engagement with participants through regular contact either telephonically or via a short-message-system.Exposure to cisplatin treatment contributed to hearing loss in females with cervical cancer, highlighting the need for ototoxicity monitoring during chemotherapy treatments. Furthermore, the results indicate that it is possible to conduct prospective cohort studies, using a multidisciplinary approach in limited-resource environments with appropriate planning and training strategies, as this study was able to achieve its aim successfully. Cervical cancer is the second most prevalent cancer, accounting for over 20% of all female cancer types in Africa . In SoutCisplatin, however, possesses ototoxic properties , where pThe impact of hearing loss on communication has serious overt ramifications on an individual’s quality of life . Consequ2) cisplatin treatment; however, it is unclear to what extent previous noise exposure, pre-existing hearing loss or the use of previous ototoxic medication may have impacted this condition.The incidence of cisplatin-associated ototoxicity ranges from 13 to 96%  and variThe longitudinal nature and degree of variability in hearing loss, after initiation of chemotherapy, required that a “fit-for-purpose” prospective study be conducted. This would allow scrutiny of audiological status prior to, during and at intervals after treatment, in the presence of confounders to exposures but also among other variables of interest. This approach also sought to reduce measurement error and avoid bias resulting from missing data associated with retrospective chart audits.The utilization of this type of study design is relatively uncommon in the audiology domain in SA, hence we embarked on a planning and feasibility phase. Here, we present the details of the feasibility study and initial results on the hearing patterns of cervical cancer patients during the course of cisplatin chemotherapy.To present details of the feasibility study and initial results on hearing patterns in cervical cancer patients receiving cisplatin chemotherapy.A prospective cohort study design was reported on.The study was conducted at a referral hospital offering tertiary services in KwaZulu-Natal (KZN), South Africa (SA), as defined in the regulations relating to categories of hospitals in SA. This hospital is also one of the main referral centers for cancer patients and houses an audiology department.The study population comprised of female patients diagnosed with cervical cancer receiving a cisplatin-based chemotherapeutic treatment regimen.Females who were 18 years or older, with an incident diagnosis of cervical cancer, commencing with the first cycle of chemotherapy were invited to participate in the study. Patients presenting with profound hearing loss at baseline assessment, or those who had previously received cisplatin chemotherapy, or had a history of medical conditions such as tuberculosis and malaria were excluded, as the treatment for such conditions often require the use of ototoxic drugs. However, since cervical cancer is considered an AIDs-defining illness, all participants were tested for their HIV status, and HIV positive women who were on ART were documented.Data was collected over a 10-month period during which 50 participants underwent audiological evaluations prior to chemotherapy initiation, at the beginning of the fourth cycle and at one-month post-treatment.Staff at the oncology department  willingly assisted with the recruitment of participants. In addition, the primary researcher checked the statistics report of the oncology department weekly to ensure that no patients were missed. Individual data collection commenced following informed consent.A detailed case history interview was conducted at each assessment to obtain information on participant’s audiological and otological status, medical conditions, medication, noise exposure, and communicative abilities related to audition. Information on the risk factors and symptoms of ototoxic hearing loss was also documented during the case history interview, in addition to patient contact details (mobile phone numbers of participants and close relatives) for the purpose of follow-up. Participants were reminded of their appointments, either telephonically or via a short message system (SMS). The monitoring of audiometry was conducted before the fourth cycle of chemotherapy, as this is generally the mid-point of the treatment regimen for patients with cervical cancer.Appropriate instructions were provided to the participants before the commencement of each audiological procedure. The audiological test, suggested for ototoxicity monitoring by the American Speech And Hearing Association  , was utiTo accommodate local prerequisites, all correspondence with participants was translated into isiZulu, which is the local language spoken by the majority of the population in KwaZulu-Natal. Therefore, information and informed consent documents, case history questionnaires, and instructions for audiological procedures were available in both English and isiZulu. An isiZulu linguist verified translations. The primary investigator was also able to converse in isiZulu, having completed a course at university. The entire audiological test took a maximum of 45 min to complete.All audiometric test results were interpreted according to the norms prescribed in the literature . On iden(a) ≥ 20 dB decrease at any one test frequency,(b) ≥10 dB decrease at any two adjacent frequencies, or.(c) loss of response at three consecutive frequencies where responses were previously obtained.On identification of a reduction in hearing ability, participants were counseled regarding treatment options such as compensatory communication strategies, as well as rehabilitation technology options, and referred to the necessary medical personnel, i.e., either the oncologist, ear-nose-throat specialist, or the psychologist. Participants were also encouraged to refrain from noisy environments, as this would exacerbate the experienced hearing loss.p < 0.05 for all statistical tests. All data were analyzed using the STATA 15 software .Feasibility issues were evaluated by considering the eligibility and accrual of participants, the capacity of the study site; follow up rates, and the results obtained in the audiological evaluation of the participants. Descriptive statistics were used to summarize demographic and clinical characteristics of the study participants, where mean and standard deviation (SD) was used for normally distributed variables and median and interquartile range (IQR) for non-normal variables. Shapiro Wilk test was used to assess normality. Categorical variables were presented using frequency and percentages and comparisons were performed using the Fisher’s exact test. The Cochran-Armitage trend test for trend in proportions was used to examine changes in case history findings over time. Ototoxic hearing loss was determined using the ASHA (1994)  guidelinApproximately five of the 30 patients diagnosed with cervical cancer at the study site each month, commenced with chemotherapy. As four participants did not meet the selection criteria, the study sample comprised 50 females. As indicated in Table n = 44) (88%), with the remaining six participants (12%) comprising of an equal distribution of the Indian/Asian and Coloured women, respectively. Fifty percent (n = 25) of the participants were diagnosed with Stage IIB cervical cancer, with stage IA2 being the least common, as diagnosed in one patient (2%). Twenty-three (46%) females were HIV positive. All participants received concomitant chemoradiation therapy. Chemotherapy consisted of cisplatin (50 mg/m2 body surface weekly for six cycles) in combination with dexamethasone (48 mg) and ondansetron (24 mg). All participants received 3 cycles of cisplatin chemotherapy (cumulative dosage of 150 mg/m2) at mid- cycle. Eight patients (16%) were found to have discontinued treatment after the third cycle, having received a cumulative cisplatin dosage of 150 mg/m2, while the remaining participants completed their cycles to varying degrees as indicated in Table The majority of participants in this cohort were black African women  (Table p = 0.42)  participants reported reduced hearing sensitivity, with the number of complaints increasing at each follow-up, although the increase was not significant (6) Table . Bilater6) Table . Reports4% particOtoscopic examination findings were normal in 48 (96%) participants bilaterally, with a tympanic membrane perforation visualized in the left ear of one participant and the right ear of another. Tympanometry was consistent with Type A tympanograms being obtained in the right ear of 49 participants (98%) and the left ear of 49 participants (98%).Clinical findings, as reflected in Table A steady increase in the number of participants with hearing loss, while not significant, is evident at each successive audiological evaluation. Sensorineural hearing loss was the most common type of hearing loss diagnosed at each audiological evaluation, with only one participant 2%) presenting with a mixed hearing loss Table . Ototoxi% presentThere is a progression of hearing loss over time, as reflected by the increase in median pure tone thresholds from the baseline assessment to each of the follow-up evaluations, especially evident from 8000 Hz to 14,000 Hz bilaterally Fig. .Fig. 3Dn = 8), Grade 2(n = 3) stages of hearing loss respectively, based on the NCI-CTCAE Grading Scale [n = 7), Grade 2 (n = 1) stages of hearing loss respectively. The Fisher’s exact test revealed no significant difference (p = 0.25), possibly attributable to the small sample size and limited follow-up.At one month post treatment follow-up, 11/23 (47.8%) HIV positive participants progressed to Grade 1 .Figure Our pilot data has demonstrated the feasibility of undertaking such a study in a resource-constrained environment, provided that the basic principles of epidemiology is adhered to and that the project is underpinned by strong intersectoral collaboration and multidisciplinary team effort. It was possible to accrue and follow up patients through study sensitization and regular contact, respectively. We observed that employing a multidisciplinary approach to the recruitment of participants was beneficial and therefore required ‘buy-in’ from the relevant healthcare personnel. This would only be possible if the healthcare personnel has been informed of the purpose of the study and the benefit to the patient population. The study site, being the main referral center for oncology services, was deemed suitable due to the high incidence of cervical cancer within a large catchment area, allowing for the generalizability of data. The hospital also has an oncology department, as well as an audiology department for robust and scientific assessment, hence site capacity is an essential evaluation parameter for such studies, given the larger scale investments that are placed into the conduct of prospective cohort studies. An audiology department away from the hospital can result in higher attrition, as patients may be too ill or may not have the funds to travel for follow up visits to additional sites. Therefore, an assessment of the environment is essential to ensure proper planning concerning patient recruitment and their time and travel management.The selection of patients with locally advanced cervical cancer was appropriate to study the impact of cisplatin on hearing loss, in this setting, given the high incidence of cervical cancer and the synchronicity of the treatment protocol. These assessment parameters allow for a higher patient population and greater probability of the inclusion criteria being met within the study period.Patient follow-up messages and appointment reminders were communicated through mobile phones via voice contact short message system (SMS). This has highlighted the growing influence of mobile phones as a tool for research and public health intervention and adds to the growing body of evidence on the value of mobile devices within health platforms , 19, 20.Our study also showed an increasing number of women with complaints of reduced hearing sensitivity and clinical hearing loss at each successive audiological evaluation.  All participants in the study presented with significant ototoxic change by the one-month post-treatment evaluation, similar to studies conducted in India , 21. TheAs is seen in ototoxic hearing loss, the findings of our feasibility study revealed sensorineural hearing loss to be most common, due to the structures of the inner ear being most susceptible to damage by cisplatin chemotherapy; with apoptotic degeneration of the hair cell in the organ of Corti being most prominent . In addiThe most common otologic symptom experienced by the study participants was tinnitus. With no national averages available in the country and 42% of participants reporting tinnitus at baseline, the current authors postulate that an increase in the stress and anxiety associated with receiving cisplatin chemotherapy, as well as thoughts about their prognosis, may have resulted in tinnitus. Hasson et al.  reportedFurthermore, 23 (46%) of the participants were HIV positive and receiving ART thus, indicating that it is not feasible to exclude patients with HIV in this cervical cancer population, as it may substantially increase the duration of recruitment. In light of this, it is essential to include information on HIV status, additional comorbidities and other potential ototoxic drugs as confounders when assessing cisplatin-associated ototoxicity. Studies indicate that some diseases such as hypoalbuminemia, anemia , renal iOur study has demonstrated that a prospective cohort study within the audiological domain is feasible in our setting, allowing for the collection of relevant medical and audiological information to investigate the hearing patterns of cervical cancer patients. This experience communicated the need for this study and may bear relevance to other resource-limited settings with a high cervical cancer burden where audiologists may wish to study hearing loss in similar cohorts, so long as the relevant expertise and support is available.


Beauveria bassiana is one of the most widely studied and used entomopathogenic fungus as biopesticide. In the biological control of pests, B. bassiana will persist in the soil after application, and will inevitably contact with earthworms, especially the epigeic earthworm species. So, what are the effects of earthworm and its epidermal mucus on the activity of B. bassiana? We employed the epigeic earthworm Eisenia fetida, B. bassiana TST05 strain, and the insect Atrijuglans hetaohei mature larvae to study the impact of earthworm epidermal mucus on the vitality and pathogenicity of B. bassiana to insect. Methods included scanning electron microscope observation, detection of spore germination, fungal extracellular enzyme activity, and infection testing to A. hetaohei. The results showed that the B. bassiana spores may attach to the cuticle of E. fetida but they could be covered by the epidermal mucus and became rough and shrunken. After treatment with the epidermal mucus, the spore germination and extracellular enzymes of B. bassiana was significantly inhibited. Inoculation of A. hetaohei larvae with a mixture of B. bassiana and mucus showed that the mucus could reduce the pathogenicity of B. bassiana to the insect, resulting in a slower disease course and lower mortality. It was concluded that the epidermal mucus of the earthworm E. fetida can inhibit the activity of B. bassiana, as well as the infectivity and pathogenicity of fungus to target insects. However, after treatment with epidermal mucus the surviving B. bassiana still had certain infectivity to insects. This is of great significance for the application of B. bassiana in biological control of pests. Beauveria bassiana, as a broad-spectrum entomopathogenic fungus, can infect more than 700 species of insects in 149 families of 15 orders and more than ten species of Acarina. It is also one of the most widely studied and used entomopathogenic fungi2. When B. bassiana infects insects, spores can adhere to the insect body surface through hydrophobic interactions. The spores absorb moisture from the insect surface to germinate and produce germ tubes. A variety of extracellular enzymes, such as chitinase and protease, are secreted from the tip of germ tubes, which can dissolve chitin and protein in the insect cuticle. The germ tubes extend continuously and penetrate into the insect cuticle and then enter the insect coelom. With the propagation of blastospores, the hyphae of B. bassiana penetrate the cuticle of insect and invade the blood cavity, infecting blood cells and internal organs. Arthrospores germinated and produced large amounts of toxins. When spores and hyphae develop to a certain number, the spores begin to decline and the hyphae increase gradually. The hyphae invade the body fat, digestive tract, Malpighian tubules, ganglion and trachea, as well as other tissues and organs, and can destroy the cells and lead to death of the insect4.B. bassiana can persist in soil and play an important role in the regulation of insect populations in soil under natural conditions5. Fungus B. bassiana is widely used as a biopesticide to control pests in farmland, orchards, vegetable fields and forests8. After application, B. bassiana can enter the soil and persist for a long time. It plays an important role in the control of pests10.Fungus 12.Earthworms are ubiquitous in farmland, orchards, vegetable gardens and woodlands and play important ecological functions in soil, such as loosening soil, improving aggregate structure, regulating soil acidity, decomposing organic matter in the soil, and improving soil fertility16. The epidermal mucus can keep the skin moist, ensure normal breathing, and play a lubricating role in the movement of earthworms. Epidermal mucus is also an important immune barrier for earthworms against pathogenic microorganisms in soil18. Earthworms can affect the species structure in soil by interacting with other organisms. It has been reported that earthworm epidermal mucus can inhibit some bacteria and affect the activities and abundance of bacteria in soil20. Mucus also has an obvious effect on the physiological function of several nematodes inhabiting soil and can regulate the quantity and distribution of these nematodes22.Earthworms can secrete mucus from their epidermal secreting cells. The epidermal mucus together with coelomic fluid discharged from the dorsal pores forms a mucus layer over their body surface. The earthworm mucus is a water-soluble mixture of low molecular weight carbohydrates, aminoacids, glycosides and glycoproteins. Among them, mucus proteins are the basic components of innate immunityB. bassiana. In the biological control of pests, B. bassiana will persist in the soil after application, and will inevitably contact with earthworms, especially the epigeic earthworm species, e.g. E. fetida. So, what are the effects of earthworm and its epidermal mucus on the activity of B. bassiana? We hypothesized that the presence of the earthworm epidermal mucus might have an antifungal effect on B. bassiana, thereby reducing their fitness. The aim of the present study was to investigate the impact of the earthworm epidermal mucus on B. bassiana biological control efficacy.However, it has not been reported whether the earthworm epidermal mucus affects the activity of E. fetida (Savigny) (Opisthopora: Lumbricidae), B. bassiana strain TST05 and the mature larvae of A. hetaohei Yang (Lepidoptera: Heliodinidae). In which, E. fetida is an epigeic species, and is the most commonly species used for composting with widespread distribution22. The strain B. bassiana TST05 is a highly pathogenic strain that was originally isolated in 2009 by our laboratory from the naturally infected overwintering larvae of Carposina sasakii (Matsumura) (Lepidoptera: Carposinidae) in the soil of apple orchards in Xiangfen County, Shanxi Province, China. On the basis of morphological identification, the strain was identified by molecular technology. After BLAST comparison, the homology of TST05 with B. bassiana  and Cordyceps bassiana (Accession No. AJ564808) in GenBank was 99%, and the support rate was 100% on the same branch of the evolutionary tree23. Therefore, the TST05 strain was identified as B. bassiana. The biological characteristics of the B. bassiana TST05 strain, pathogenicity to host insects, persistence in soil, and compatibility with chemical insecticides were studied in previous researches26. At the same time, the strain was deposited in the China General Microbial Species Conservation and Management Center  under storage number CGMCC4526. The mature larvae of A. hetaohei Yang (Lepidoptera: Heliodinidae) were selected as the target insects infected by B. bassiana strain TST05. It is an important pest of walnut fruit in northern China. The insect produces one generation a year, and its larvae bore into walnut fruit to feed on the pulp, which makes the walnut fruit lose its commercial value. The newly hatched larvae bore into the fruit in May and develop to maturity in late August. They drill out of the fruit and fall into the soil layer to live through the winter and cocoon and pupate. In the early summer of the next year, the adults emerge and bore out of the soil and then mate and lay their eggs in walnut trees. Under natural conditions, larvae are often infected by entomogenous fungi such as B. bassiana in soil. The biological control of A. hetaohei using B. bassiana and other entomopathogenic fungi has attracted much attention27. An infection test of B. bassiana strain TST05 on A. hetaohei and the application of a strain preparation in walnut orchard soil were carried out in prior study to control the mature larvae, and high infection mortality was achieved28. Therefore, the mature larvae of A. hetaohei were selected as the target insects infected by B. bassiana strain TST05 in this study. In addition, after entering the soil, the mature larvae of A. hetaohei can exist together with earthworm E. fetida and B. bassiana TST05 strain for 8–9 months. Thus, it is a typical insect in the walnut orchard soil environment.We employed a model system composed of earthworm B. bassiana in the activity and pathogenicity to the insects. It is significant for the application of B. bassiana as a biopesticide in the field.This study provides a reference for understanding the impact of earthworm epidermal mucus to B. bassiana spores often adhere to the intersegmental furrows between the segments, annulus on the segments, grid-like structures between the annuli, and those around the stomata of E. fetida .After incubating with solutions of epidermal mucus, the colonies of cus Fig. . In the ate Fig. A, with 1ate Fig. . In contsed Fig. B–F. In t6% Figs. B, 3. In 5% Figs. C, 3. The5% Figs. D, 3. An es Figs. E,F, 3. SE. fetida was added to the culture medium to assess whether the E. fetida epidermal mucus would affect the extracellular enzyme activity of B. bassiana. The results showed that the enzymatic activities of the 4 extracellular enzymes studied significantly changed after treatment , the fungal protease  enzyme , the enzymatic activities of the subtilisin-like protease, lipase, chitinase and NAG remained at very low levels , compared with the other two groups, the activities of 4 extracellular enzymes showed different trends. During days 1–4 after inoculation, the enzymatic activities of the 4 extracellular enzymes remained at very low levels , the cumulative corrected mortality rate was 11.236 ± 2.570% and 47.126 ± 3.320%, respectively. After 5 d, the mortality rate of larvae in the treatment group increased significantly. At 7 d, 10 d, and 14 d, the cumulative corrected mortality of larvae was 10.588 ± 4.342%, 45.238 ± 4.025%, and 67.901 ± 3.782%, respectively, while that in the CK group was 74.118 ± 5.263%, 94.048 ± 4.166%, and 100%, respectively , and the upper epidermis includes the wax protecting layer, wax layer and cuticle seminal layer. The insect epidermis consists of an epicuticle and procuticle. The epicuticle includes the cement layer, wax layer and cuticulin layer from external to internal, and the procuticle is mainly composed of chitin and protein. Additionally, extracellular protease, chitinase and lipase are secreted by B. bassiana to degrade protein, chitin and ester of the insect cuticle. The activity of extracellular enzymes of B. bassiana is usually considered an important index to detect its infectivity to host insects41. In fact, these four groups of extracellular enzymes secreted by B. bassiana belonging to extracellular proteases, chitinases and lipases which, are usually considered to be secreted by B. bassiana in degrading protein, chitin and ester of the insect cuticle. The exact number of enzymes in each of four groups could be further estimated by mining the known genomes of B. bassiana species. This problem may need further study.In the process of E. fetida epidermal mucus lyophilized powder was added to liquid medium every day in the treatment group EW, and the E. fetida epidermal mucus lyophilized powder was only added once at the beginning of culture in the treatment group EO. It was found that during incubation of B. bassiana, the epidermal mucus of the earthworm E. fetida did have a significant inhibitive effect on the 4 fungal extracellular enzymes , and the inhibitive effect could remain for a period of time. Therefore, it is suggested that the epidermal mucus of E. fetida can inhibit B. bassiana not only for spore germination but also for extracellular enzyme secretion. However, if B. bassiana is no longer exposed to the newly secreted E. fetida epidermal mucus, the inhibition effect will gradually decrease.In this study, B. bassiana in soil in which the earthworm Lumbricus terrestris (L.) has been present could still infect Galleria mellonella (L.) larvae. However, the specific effect of the earthworm epidermal mucus on B. bassiana infection was not studied. To verify whether the E. fetida epidermal mucus had an effect on the pathogenicity of B. bassiana, the larvae of A. hetaohei were inoculated with B. bassiana spores that were treated with the E. fetida epidermal mucus in advance. The results showed that the E. fetida epidermal mucus could truly reduce the pathogenicity of B. bassiana to the insect. It could result in a slower disease course and a lower mortality of the larvae during infection. The practical data showed that within 5 d, the mortality rate of the larvae infected by B. bassiana in the group treated with E. fetida epidermal mucus was much lower than that of the CK group, but it increased rapidly after 5 d. The mortality rate of the treatment group increased from 3.45% on the 7th day to 41.67% on the 10th day , but it reached 91.67 ± 3.85% in the CK group. The mortality rate of the treatment group at 14 d was 65.43 ± 6.78%, while it reached 100% in the CK group. Moreover, the median lethal time of B. bassiana to the larvae in the group treated with the E. fetida epidermal mucus was 10.322 d in the treatment group, but it was 5.496 d in the CK group. Therefore, although the pathogenicity of B. bassiana to the insect A. hetaohei was affected by the epidermal mucus of the earthworm E. fetida and was reduced in infectivity to the insect, those B. bassiana spores that survived in the treatment with E. fetida epidermal mucus still maintained certain infectivity to the insect. In addition, the inhibitory effect of the E. fetida epidermal mucus on B. bassiana can gradually be relieved after 5–7 d with loss of the active component from the E. fetida epidermal mucus.Shapiro Ilan and Brown (2013) found that E. fetida and fungus B. bassiana indicated the following conclusion: the epidermal mucus secreted by E. fetida has a significant inhibitory affect on the vitality and pathogenicity of B. bassiana. The inhibitive effect includes limiting fungal spore germination, reducing fungal extracellular enzyme secretion and activity, and weakening fungal infectivity and pathogenicity, which caused a slower infection and less mortality of the target insect. These inhibitory effects are interrelated with the concentration and extension time of the epidermal mucus of E. fetida. Nevertheless, those B. bassiana spores surviving after treatment with E. fetida epidermal mucus still maintain a certain infectivity to the target insect. When B. bassiana is used as a biological insecticide, the application methods include spraying, dusting or directly applying into the soil. Whichever method is used, it will eventually stay in the soil and contact with earthworms. Previous studies did not consider the effect of earthworm epidermal mucus on B. bassiana. Our study found that the earthworm epidermal mucus has a certain inhibitory effect on B. bassiana, including inhibiting spore germination and reducing the pathogenicity to insects. This finding is very important for applying B. bassiana in the biological control of insects. However, it is necessary to study the antifungal mechanism of the epidermal mucus of the earthworm E. fetida to B. bassiana.As mentioned above, the experiment using the epidermal mucus of the earthworm Eisenia andrei was up-regulated after treatment with Bacillus subtilis and Escherichia coli42. It was also reported that the expression of cellulase, Lumbrokinase, lysozyme, HSP70 and other immune-related proteins in E. fetida were up-regulated after treatment with E. coli43. Under the stimulation of Saccharomyces cerevisiae, the expression of pattern recognition receptor CCF (coelomic cytolytic factor) and lysozyme was up-regulated in E. andrei44. Earthworm epidermal mucus is the first defense line of its innate immunity. Earthworms epidermal will secrete a large amount of mucus in response to a stimulus, and form a mucus film wrapped in the surface of the earthworm, which has a protective effect on the earthworm22. However, it is not reported whether the genes related to innate immunity and mucus secretion of earthworms will change their expression after the earthworms are treated with B. bassiana. Earthworm epidermal mucus is also a complex of many components and the secretion of mucus should be involved by many genes, so it is a subject worthy of further study in the future.Earthworms live in an environment with a variety of microorganisms. In order to cope with changing environmental conditions, earthworms require highly responsive stress mechanisms. It has been reported that the expression of immune-related earthworm cellulase in E. fetida were obtained from the earthworm breeding base in Baiyangdian, Hebei, China and raised in a laboratory at 20 °C with 70% soil humidity. Adult clitellate earthworms of similar body size were selected for this study.Earthworm B. bassiana TST05 strain was cultivated in the laboratory at 25 °C with 75% relative humidity (RH), and fungal conidia were collected for the study. Two media were used in the experiments described below: (1) B. bassiana rejuvenation: Potatoes 200 g, Glucose 20 g , Agar 20 g , cuticle powder of target insects 5 g, Distilled water 1000 mL, pH value of the natural45. (2) Potato Dextroxa Agar (PDA): Potatoes 200 g, Glucose 20 g , Agar 20 g , Distilled water 1000 mL, pH value of the natural.The A. hetaohei Yang (Lepidoptera: Heliodinidae) were selected as the target insects infected by B. bassiana strain TST05 in this study. Before the experiment, walnut worm fruit was collected in late August from an experimental walnut orchard in Yuxian County, Shanxi Province, China, and then, the walnut worm fruit was placed in the laboratory in the shade. When the mature larvae naturally drilled out of the fruit, they were collected and placed in a culture dish covered with a wet filter for infection study.The mature larvae of B. bassiana spores were placed in sterilized distilled water containing 0.1% Tween 80 and the concentration was 1.0 × 107 spores/mL. Spore concentration was counted under microscope by using cell counting plate. The immersion method was used for earthworm infection. Earthworm E. fetida sample individuals were steeped for 10 s into the spore suspension of B. bassiana and then maintained at a temperature of 15 ℃ for 10 min. Then, earthworms were transferred in a beaker (500 mL) lined with wet filter paper. Samples were collected at 1 h, 3 d and 7 d and fixed with 2.5% glutaraldehyde solution at 4 °C for 48 h. Then, the earthworm samples were rinsed thrice with 0.2 M phosphate buffer and incubated for 1.5 h in 1% (v/v) osmium tetroxide in the same buffer at 4 °C. Then, the samples were washed with ddH2O and dehydrated in a series of acetone solutions, which was replaced by liquid carbon dioxide. Finally, the samples were dried using an EMS 850 critical point drying apparatus  and treated with gold spray. The gold-sprayed samples were observed and photographed using a scanning electron microscope (SEM) .The E. fetida individuals were stored in a sterile beaker (500 mL) with sterilized distilled water. After 30 min of incubation, the epidermal mucus secreted by the earthworms was collected with sterilized distilled water. Then, a filter membrane was used to remove the bacteria, and the water was removed from the epidermal mucus using a freeze-drying technique. The dry matter of the epidermal mucus of E. fetida was prepared into 1.25 mg/mL, 2.50 mg/mL, 5.00 mg/mL, 10.00 mg/mL, and 20.00 mg/mL mucus solutions using sterilized distilled water. Sterile distilled water was used instead of the earthworm epidermal mucus solution in the control group. The treatment of each concentration was repeated 3 times.The B. bassiana spores (200 spores/mL) was mixed with different concentrations of the mucus solutions in a 1:9 ratio. After incubation at 4 °C for 2 h, 1 mL of the mixture was smeared onto PDA medium and incubated at 25 °C for 7 d. The fungal colonies of B. bassiana were observed and counted. The experiment was repeated 5 times in each group.A suspension of 2PO4, 0.6 g/L MgSO4, 0.5 g/L KCl, 1 mg/L FeSO4·7H2O, 1 mg/L ZnSO4·7H2O, and 7.5 g/L insect cuticle powder) were sterilized by autoclaving. Then, 1 mL of the spore suspension (108 spores/mL) of B. bassiana and 9 mL of the epidermal mucus solution (2.5 mg/mL) of E. fetida were mixed and inoculated into the medium. Then, the triangle bottles were put into an oscillator and incubated at 25 °C for 8 d. During the culture process, the treatment group EW was supplemented with 10 mg earthworm mucus lyophilized powder every day, but the treatment group EO was no longer given epidermal mucus lyophilized powder. In the control group (CK), only spore suspension was added, and the earthworm epidermal mucus solution was replaced by sterile distilled water. The experiment was repeated 5 times in each group.Triangle bottles with 40 mL of the liquid medium 2-Pro-Phe-pNA to produce 1 μg nitroaniline per minute.Subtilisin-like protease assay: The method reported by St. Leger was employed (1987)47. One unit of lipase activity was defined as the amount of enzyme that catalyzes the decomposition of fat to produce 1 μg p-nitrophenol per minute.Lipase assay: The method reported by Silva was employedN-acetylglucosamine per hour.Chitinase assay: Chitinase was determined according to the instructions of the Chitinase Kit . One unit of chitinase activity was defined as the amount of enzyme that catalyzes the decomposition of chitin to 1 μmol N-acetylglucosaminidase Kit (A031-1-1 Nanjing Jiancheng Bioengineering Institute). One unit of NAG activity was defined as the amount of enzyme that catalyzes the hydrolysis of 1 μmol substrate per minute.NAG assay: NAG was determined according to the instructions of the β-B. bassiana spores (107 spores/mL); (4) a suspension of B. bassiana spores (108 spores/mL) mixed with earthworm epidermal mucus (2.5 mg/mL) in a 1:9 ratio. Each treatment was repeated 5 times.Four treatments were set up in the experiment: (1) sterilized distilled water only; (2) sterilized distilled water mixed with earthworm epidermal mucus (2.5 mg/mL) in a 1:9 ratio; (3) a suspension of A. hetaohei were employed as infection insects. The larvae were soaked in the mixture or sterilized distilled water for 5 s and then placed in a Petri dish lined with wet filter paper and absorbent cotton at the bottom. Forty larvae were placed in each Petri dish, and each experiment was repeated 3 times. The larvae were placed in a 25 °C incubator, the activity and surface characteristics of larvae were observed every day, and the quantity of dead larvae was counted. After 15 days of observation, the corrected mortality was calculated.The larvae of Data were analyzed using the SPSS 21.0 statistical software package. ANOVA and Tukey's HSD were performed for statistical analyses.


Electrophysiological investigations reveal a great deal about the organization and function of the retina. In particular, investigations of explanted retinas with multi electrode arrays are widely used for basic and applied research purposes, offering high-resolution and detailed information about connectivity and structure. Low-resolution, non-invasive approaches are also widely used. Owing to its delicate nature, high-resolution electrophysiological investigations of the intact retina until now are sparse. In this Mini Review, we discuss progress, challenges and opportunities for electrode arrays suitable for high-resolution, multisite electrophysiological interfacing with the intact retina. In particular, existing gaps in achieving bi-directional electrophysiological investigation of the intact retina are discussed. Non-invasive electrophysiological interfaces for the retina are important in the study of the retina and the visual system. Non-invasive electrophysiological investigations are widely used both for medical and research purposes . Primariex vivo investigation of the retina is another important example of the utility of electrophysiology in the study of the retina. It is widely applied to understand neural processes in retina development, function, and degeneration. Spontaneous retinal waves, as an example, is a electrophysiological phenomena of the developing retina  have spontaneous bursts at 100–240 ms intervals, similar to that observed in the developing retina  responses to light stimulation, and retina cell (ganglion or bipolar) responses to electrical stimulation, to name just two examples , with properties similar to those observed in the explanted retina.Soft devices suitable for retinal stimulation are also emerging. For example, a stimulating photo-capacitor on soft and thin (20 mm) polyurethane (PU) film was recently demonstrated in Recently, ex vivo conditions may be distorted. For example, identifying stimulation parameters for retinal implant performance optimization may depend on retina metabolic conditions  limits the ability to carry out such investigations on patients. For example, desensitization, the process in which visual perception is fading has been debated . Ex vivoecessary . Recordiecessary , in partIt is important to note that epi-retinal configurations have so far showed limited performances, and recent vision restoration strategies focus on sub-retinal or cortical stimulation. A prosthesis showing great results include subretinal implant PRIMA (Pixium Vision) and cortical prosthesis ORION  . NeverthThe study of brain abnormalities through abnormal retina function is a relatively recent field which may also benefit from better interfacing with the intact retina. Non-invasive approaches have revealed several interesting insights. Multiple ERG studies showed retinal functionality anomalies  in schizophrenia, autism, bipolar disorder, multiple sclerosis and other psychiatric and neurodevelopmental disorders .Another interesting domain which can gain from high-resolution electrophysiological interfacing with the retina is transorbital altering current stimulation (ACS). It was suggested that transcranial or transorbital ACS could be used to treat various psychiatric disorders or optic nerve conditions, respectively . High-reex vivo. Ganglion cells with intrinsic light sensitivity can be optically stimulated and electrically measured. Studies investigating the effect of circadian disruption and ipRGC activity would provide more knowledge and suggest new treatment methods.The study of the circadian clock is yet another example for the possible benefits of bidirectional electrophysiology from the intact retina. There are indications that circadian clock disruption may be a factor in age-related macular degeneration incidence, behavioral health, and psychiatric problems . Blue liex vivo model of the retina remain. For example, bursts occurred more frequently in vivo than in ex vivo retinal investigations (ex vivo conditions? The study of spontaneous waves in the intact retina will allow the investigation under more neutral conditions. Spontaneous waves or retina hyperactivity are also known to appear in retina degeneration. Whether and how such retinal activity affects retinal degeneration is interesting and can impact retina implant technology (Finally, spontaneous waves in the retina have been studied extensively, however, questions regarding the validity of igations . Is thischnology . Walter Reports on flexible and soft neural electrodes for deep brain stimulation, sciatic, tibial, vagus nerve stimulation are abundant . Under tin vivo high-resolution electrophysiological studies of the retina, much of what we know about the retina electrical activity is based on the explanted retina model. Explanted retinas of different animal models can be readily investigated ex vivo using high-resolution electrophysiology. In particular, multi electrode arrays are commonly used to study retina electrical activity during the developmental stages, degenerative diseases, or to investigate retina responses to electrical or optical stimulation, to name just a few examples. Recent progress toward more stealthy neural probes establishes important first steps toward the wider adoption of soft retinal interfaces. Finally, better devices that are suitable for bi-directional retinal interfacing will contribute to improved technology that can be used far and beyond retinal interfacing.Owing to the challenging intra-ocular position and delicate nature of the retina, high-resolution devices for bi-directional recordings and stimulation from the intact retina are presently unavailable. As it was reviewed above, some soft electrodes with high resolution are beginning to meet the requirements needed for high-resolution electrophysiology in the intact retina. Moreover, soft neural interfaces that target the retina provide exciting opportunities in the investigation of retina structure and function. Such devices may play a role in a wide range of applications, ranging from basic investigations, to better understanding of artificial retina stimulation, to closed loop electrical stimulation of the retina.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.