Background: Menière's disease (MD) is an inner ear disorder characterized by recurrent episodes of spontaneous vertigo, unilateral low-frequency sensorineural hearing loss, tinnitus, and aural fullness. Current diagnosis still often has to rely on subjective and audiometric criteria only, although endolymphatic hydrops is recognized as the pathophysiological substrate of the disease, having been demonstrated in anatomical pathological studies and by magnetic resonance (MRI). The modiolus has a close functional and anatomical relationship with the cochlear nerve and membranous labyrinth and can be evaluated with MRI but no data exist on the modiolar size in MD.Purpose: Our purpose is to examine the following hypothesis. Is cochlear modiolus smaller in symptomatic ears in MD?Methods: We used a retrospective 3 Tesla MR study (heavily T2-weighted 3D fast asymmetric spin-echo images and 0.5 mm slice thickness) comparing the mean modiolar area (MMA) in the index and best ears of eight patients with definite MD based on audiometric data. The obtained MMA values were compared against the audiometric data and the presence of vestibular endolymphatic hydrops.Results: No differences were seen in MMA between best and worst ears. Ears with a pure tone average (PTA) ≥25 dB and more pronounced endolymphatic hydrops showed lower MMA . Two patients with extreme endolymphatic hydrops showed a noteworthy ipsilateral decrease in the cochlear modiolus area.Conclusion: No differences were seen in MMA between best and worst ears in definite MD. Worse hearing function (PTA ≥ 25dB) and more pronounced endolymphatic hydrops seem to be associated with lower MMA. This might be related to bone remodeling as a consequence of endolymphatic hydrops. Further research is needed to corroborate and explore these findings. MD is a chronic disease with a prevalence of 200–500 per 100,000 individuals , using an 8-channel head coil. Thin-section heavily T2-weighted 3D fast spin-echo, TR 1500/TE 200, echo train length 61, field of view 150, slice thickness 0.5 mm, axial slab matrix 256, voxel size 0.5 AP/0.7 RL/0.5 FH and scan time 4.34 min.Thin section MR images of 16 ears of eight patients with definite MD were obtained. Patients were all recruited from the outpatient otoneurological department of Hospital Santa Maria (Lisbon). They were clinically evaluated by one senior (LL) and one junior (TE) otoneurologist who classified the patients with definite MD according to the clinical Barany Society criteria  and defiIn our study, we used a volumetric T2 acquisition with 0.5 mm thickness. Two neuroradiologists, blinded to the clinical diagnosis, independently evaluated the area of the cochlear modiolus on the MR console, using multiplanar reformatting. This area was measured in the axial slice in which the cochlear modiolus was visualized at its maximum size from the thin section T2-weighted images (midmodiolar level). A region of interest was manually drawn and measured by each radiologist twice: the measured area outlined the low signal trapezoidal or triangular shape at the axis of the basal turn of the cochlea, or the middle and basal turn, excluding the thin free part of the osseous spiral lamina and interscalar septum , 14. TheAdditionally, the vestibular endolymphatic space was evaluated, based on MR images obtained with HYDROPS protocol 4 h after intravenous injection of a single dose of gadolinium, as previously described .The area of vestibular endolymphatic space and the area of the total membranous labyrinth was manually traced by a radiologist, using multiplanar reformatting, according to the plane of the lateral semicircular canal (including the ampulla in the measurement), using volumetric acquisitions . The final value was obtained as the percentage of endolymphatic area to total labyrinth area. We couldn't quantify cochlear endolymphatic space because of image quality limitations. All image evaluation procedures were performed by researchers blinded to the clinical data of the patients.U test. A p-value of 0.05 was considered statistically significant. Interrater reliability was also assessed using the intraclass correlation coefficient (ICC).The two groups were compared using the Mann-Whitney Cochlear modiolus area, PTA, and vestibular endolymphatic space were registered in Three MD patients were male and five were female. Ages ranged from 47 to 52 in MD group (mean age of 48). Disease duration ranged from 2 to 24 years (mean duration of 7 years). All patients had spontaneous vertigo attacks lasting from 20 min to 12 h and fluctuating symptoms; number of vertigo attacks ranged from 2 to 10 episodes/year; and postural instability between attacks was described by two patients . Pure to2 (3.03 to 3.94 mm2). Interrater reliability was good: ICC = 0.90, with a 95% confidence interval (0.59–0.97). We did not find differences of MMA between “index ears” (3.51 mm2) and “best ears” groups (3.52 mm2) (p = 0.3320).The MMA in this MD population was 3.52 mm.52 mm2) , but earVestibular endolymphatic hydrops was present in 88% of patients with definite MD (7/8 patients). Vestibular endolymphatic hydrops was found in 11/16 ears (69%) according to Barath's criteria  cut-off , but this was more evident in patients one and two, which interestingly had more extreme endolymphatic hydrops and a higher number of acute episodes and a relatively recent diagnosis of the disease , Table 12 if we assume it has a triangular shape, which is not always true. Naganawa et al. first measured cochlear modiolus with MRI in 1999 in asymptomatic healthy volunteers  (instead of 0.8 mm used by Naganawa) in order to decrease the partial volume effect. Naganawa et al. , however2 (3.03–3.94 mm2), which seems to be lower than in the healthy population. However, we cannot make definite conclusions regarding this and future prospective controlled studies can confirm this finding with greater confidence: we will expect a global decrease in modiolus size in index and non-index ears in MD compared to the healthy asymptomatic control group.A volumetric measurement should be more precise, but we would need higher-quality images. As this was a retrospective study, we did not have data from a group of healthy volunteers (it would be difficult to reproduce the same conditions in the new available MR scanner). For this reason, we can only compare our MD data with healthy volunteers data from previous histological , 20 and In this study, we did not find significant statistical differences in MMA between “asymptomatic” and index ears in MD. Maybe this can be related to the fact that the “asymptomatic” or “best” ear is not truly healthy in some patients as we can see from the PTA records. This explains why a slight decrease in MMA is seen in ears with PTA higher than 25 dB or in ears with larger endolymphatic spaces. This aspect was more evident in two particular patients (patients one and two) even in relatively short-term disease . Our endolymphatic space areas refer to the vestibular component only because of image quality limitations in cochlear endolymphatic space.Increased hydrostatic pressure can explain these findings, based on increased endolymph fluid and decreased microvascular supply (stria vascularis), causing bone remodeling and demineralization, respectively. This aspect has been demonstrated in previous histological studies , 20 and In fact, it seems MD is an entity whose clinical criteria have some inherent diagnostic subjectivity based on symptoms only, and MD seems to include a wide range of distinct etiologies, being difficult to unify in a single entity. Endolymphatic hydrops seems to be present in most patients with a clinical diagnosis but also in other patients with an incomplete clinical picture of MD or secondary to other etiologies , which has lead to the concept of Hydropic Ear Disease, encompassing all typical and atypical variants of MD in one logical framework –23. For In the last years, a lot has been published regarding imaging in MD, mainly related to endolymphatic hydrops, which is the pathophysiological correlate of the disease. Endolymphatic hydrops is present in the greater majority of MD patients but can be also be found in other entities. Recently other additional imaging signs have been shown to increase the specificity of endolymphatic hydrops in MD , such as the increased intensity of the perilymphatic space after gadolinium injection in the index ear , which rThe modiolus area is not significantly different between the index and best ears in MD, which can be explained by the tendency of bilateral affection of the disease and might reflect its systemic nature. However, the modiolus area seems to be smaller in ears with extreme endolymphatic hydrops, causing worse hearing function in patients with clinical criteria for definite MD, which can be justified by increased hydrostatic pressure resulting in bone remodeling of cochlear modiolus and cochlear nerve endings damage. MR imaging of the cochlear modiolus deserves more investigation, however, especially in patients with endolymphatic hydrops, with or without fulfillment of traditional clinical criteria for MD (termed “hydropic disease”) , 22. AssThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Comissão de Ética da Faculdade de Medicina da Universidade de Lisboa. The patients/participants provided their written informed consent to participate in this study.RS, CG, TE, JC, and LL contributed to conception and design of the study. RS and CG evaluated the MR images. TE and LL made the clinical evaluation. RS and CG organized the database. CG performed the statistical analysis. RS wrote the first draft of the manuscript. RS, CG, and TE wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mucilage is a gelatinous high-molecular-weight substance produced by almost all plants, serving numerous functions for plant and soil. To date, research has mainly focused on hydraulic and physical functions of mucilage in the rhizosphere. Studies on the relevance of mucilage as a microbial habitat are scarce. Extracellular polymeric substances (EPS) are similarly gelatinous high-molecular-weight substances produced by microorganisms. EPS support the establishment of microbial assemblages in soils, mainly through providing a moist environment, a protective barrier, and serving as carbon and nutrient sources. We propose that mucilage shares physical and chemical properties with EPS, functioning similarly as a biofilm matrix covering a large extent of the rhizosphere. Our analyses found no evidence of consistent differences in viscosity and surface tension between EPS and mucilage, these being important physical properties. With regard to chemical composition, polysaccharide, protein, neutral monosaccharide, and uronic acid composition also showed no consistent differences between these biogels. Our analyses and literature review suggest that all major functions known for EPS and required for biofilm formation are also provided by mucilage, offering a protected habitat optimized for nutrient mobilization. Mucilage enables high rhizo-microbial abundance and activity by functioning as carbon and nutrient source. We suggest that the role of mucilage as a biofilm matrix has been underestimated, and should be considered in conceptual models of the rhizosphere. Zea mays L.) crown root mucilage harbors nitrogen-fixing bacteria, which contribute to the fixation of a considerable amount of the plant’s nitrogen requirement  and surface tension (N m–1) values. A list of plant and microbial species that produced the biogel, accompanied by their references, has been provided in We considered some criteria for selection of the data. For the physical properties, the viscosity values had been measured at a solute concentration of 0.5 mg mlTo calculate the mucilage spatial and temporal distribution at a given exudation rate around a growing model root, we assumed a root-soil system with the following parameters: Soil porosity = 50%; crown root diameter = 3.3 mm (unpublished data of maize); mucilage exudation rate = 1.41 mg dry weight per day and root tip (unpublished data of the same maize plants); maximum hydration ratio of mucilage = 425:1 , assuming a 39% water saturation upon exudation at the root tip and a rapid saturation to 100% within 6 h ; root elCin) was assumed to produce bacterial biomass under carbon limitation. The growth and biomass yield of several rhizosphere bacteria using glucose are comparable to the growth achieved by mucilage as a sole carbon source (fc = 0.77) to be available for bacterial consumption . The upper bound on mucilage-derived carbon (Cin) that can be allocated to produce bacterial cell biomass (MB) was obtained by considering an average carbon use efficiency (CUE) for a range of carbon sources (MB = cf × cw × CUE × Cin). We assumed that maintenance costs were negligible and all cell biomass could be produced within a day. For the calculation presented here, we used an average CUE of 0.6 based on genome-scale metabolic predictions (Ncell) that could feed on degraded mucilage, we divided the cell biomass carbon by an average bacterial cell carbon mass (Mcell) of 10 fg C per cell (cellN = B/McellM)  .MEPS) by a given bacterial abundance by using an EPS yield per unit of cellular biomass (EPSM = BM × YEPS with EPSY = 10 mg g–1)  . Our modt-test was used to test for significant differences between mucilage and EPS in terms of the investigated properties at the significance level of 0.05. All charts were designed using SigmaPlot 14.0 .All data were analyzed by IBM SPSS Statistics for Windows, version 25 . The data were tested for homogeneity of variance and normality by Levene’s test and Shapiro–Wilk test, respectively, and transformed logarithmically if they did not fulfill these prerequisites. The Independent Samples –1 and 0.051 N m–1, respectively.The viscosity of mucilage and EPS did not differ significantly . AveragePolysaccharides were the major chemical constituent of both biogels  and did not significantly differ between them . The samSix out of nine studied monomers of the biogels’ polysaccharide backbone did not significantly differ in proportion between mucilage and EPS, namely galactose , fucose , glucose , rhamnose , xylose , and glucuronic acid . In contrast, mannose  was significantly higher in EPS than in mucilage (nearly fivefold higher), whereas arabinose  and galacturonic acid  had higher proportions  in mucilage than in EPS .–1 soil. The kinetic of water saturation and swelling is rapid with an average 6 h until the mucilage of root tips reaches its constant volume. Therefore, 9.2 mm above the root tip, the mucilage is fully hydrated, reaching its final radial extent of 2.05 mm. By swelling, the content decreases to 0.8 mg g–1 soil, a value hardly changing by decomposition along the daily grown segment of 30 mm. Assuming a linear decomposition rate of 50% in 7 days, mucilage is only half decayed at a distance 21 cm above the root tip.Based on simplified assumptions of root growth, exudation rate, decomposition rate, hydration ratio, and mucilage expansion into the soil, a simple model for the size and extent of a potential “mucilage biofilm” was developed . The axiin_rc) is 5 μg d–1, and (ii) the 28.35 mm root zone above the root tip (grown within 1 day) where the mucilage concentration is reduced by gel swelling and degradation (Cinez = 122 μg d–1). The high C concentrations at the root tip allow for a maximum number of bacterial cells growing on the basis of mucilage C consumption, which is in the order of 109 cells per day. In contrast, lower C concentrations at the 28.35 mm zone above the root tip resulted in only 3 × 1010 bacterial cells grown per day. Despite its 25 times larger volume, the 28.35 mm root above the root tip can only host around 10 times more bacterial cells than the small soil volume surrounding the 1.65 mm of the root tip. The bacterial cells could potentially produce around 0.01 μg EPS per day at the root tip and 0.29 μg EPS per day within the 28.35 mm zone of daily growth. Assuming the above given mucilage exudation of 1.41 mg d–1 and root tip with a 50% decomposition in 7 days , 101 μg of mucilage covering the root gets decomposed and replaced by 0.3 μg of EPS per day, assuming mucilage as the sole C source. This leads to a relatively small but continuous modification and thinning of the biofilm along the root axis.We consider that two different zones around the root receive degradable carbon at varying rates: (i) the zone within 1.65 mm from the root tip where the estimated total mucilage C exudation rate , and lipids. However, the proportion of these constituents is low . EPS conExtracellular polymeric substances eDNA increases the structural stability of biofilms, functions as an important agent of microbial aggregation, and acts as an intercellular connector . MucilagLipids play a part in the hydrophobicity of EPS and help microorganisms adhere to waxy, plastic , and pyrite surfaces . Similar2+ bridges in mucilage, which connect the uronic acids and therefore increase the mucilage stability  released at the zone of maximal root exudation also buffer the proton exudation from the root, these compounds are of low molecular weight and are consequently rapidly decomposed. In contrast, galacturonic acid with a logarithmic acid dissociation constant (pKa) of 3.5 might therefore more efficiently and over a larger zone of the mucilage-covered rhizosphere fulfill this buffering function, at least in very acidic soils. This might be a mechanism contributing to avoiding a very strong rhizosphere acidification and thus a limitation of microbial life and activity .In summary, we found a remarkably similar chemical composition of mucilage and EPS, which supports the contention that mucilage can function as a biofilm matrix. Although the individual role of each chemical mucilage component and its impact on microbial life has not yet been unraveled, it is unlikely that the limited deviation in chemical composition between mucilage and EPS would significantly diminish the potential of mucilage to function as a biofilm matrix.via catabolism and oxidized to gain energy . This suggests that low-molecular-weight exudates may play a minor role as C substrate for EPS production, but more importantly, that their function as a microbial C source might have been overestimated compared to mucilage C.The contribution of EPS to the root-covering biogel was already suggested by d–1 cm–1 . Even asd–1 cm–1 , this da10 cells per cm3 mucilage-affected rhizosphere volume, or 109 bacterial cells per g mucilage-affected rhizosphere soil  can be found in the article/MN and MD proposed the idea. MN performed the literature review and comparative analyses, and wrote the manuscript draft. SB developed the bacterial abundance and EPS production model. PB and AC developed the mucilage exudation model. KM-J shared ideas and scientifically enriched the text. MD supervised the project. All authors read the manuscript draft, commented on it, and confirmed it before submission.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.


Steinernema feltiae from Poland are described. New S. feltiae isolates from Poland showed close genetic similarity to other isolates of this species and exhibited a high reproductive rate and invasive capacity compared to the commercial biopesticide.Entomopathogenic nematodes (Steinernematidae) are obligate insect parasites and are used for insect pest control, particularly on amenity grassland and in horticulture. Extensive surveys have been conducted across the globe to isolate locally adapted entomopathogenic nematodes species. The biological activity and morphology of three new isolates of Steinernema feltiae from Łoniów were the largest. The smallest infective juveniles were found in the isolate from Oblasy, and the smallest males in the isolate from Danków. In Poland, new field isolates showed close genetic similarity to other S. feltiae isolates. The research showed that the field isolates from Poland had greater infectivity and rate of reproduction compared with nematodes from the commercial biopesticide. The findings indicate the potential use of field S. feltiae isolates from Poland  to develop new biopesticide products.Insect trap studies were carried out to determine the presence of entomopathogenic nematodes (EPN) from the family Steinernematidae in the soils of Poland and to compare the biological activities of field nematode isolates with nematodes from commercial biopesticide. The fauna of these organisms in central Poland is poorly studied in both taxonomic and biological terms. Tilled soils representative of this region were sampled from cultivated fields. EPN were isolated from soil samples under laboratory conditions and identified using a key for species identification and molecular analysis. Basic morphometric parameters of infective juveniles and adult males of the first generation were determined. The research showed that males and infective juveniles  The abundance and activity of soil fauna are largely dependent on the level of human activity within a given sampling area. Species diversity usually declines with increasing land development, resulting in a detrimental effect on biogeochemical cycles in ecosystems, disrupting the natural food webs and increasing the populations of plant pests. In crops, pest control may be particularly difficult in some areas due to, among other things, regulatory restrictions for applying plant protection chemicals. Therefore, the development of biological control methods to manage insect pests, using natural enemies, is an effective alternative strategy by which to achieve pest management, but without damage to the environment. The widely used biocontrol factors include: bacteria, viruses, fungi, entomophagous insects and entomopathogenic nematodes (EPN) ,6,7,8,9.Research into new isolates of EPN indicates that they can differ greatly in terms of invasiveness and survival, and tolerance to various environmental factors ,14,15,16Xenorhabdus (in Steinernema spp.) and Photorhabdus (in Heterorhabditis spp.), e.g., X. bovienii Akhurst, 1983, and P. luminescens Thomas et Poinar, 1979 [EPN are natural inhabitants of the soil environment ,19,20,21ar, 1979 , althougar, 1979 ,24. The ar, 1979 ,25.S. feltiae Filipjev, 1934, is a cold-adapted species, infecting hosts within the range 8–30 °C and reproducing at 10–25 °C [The biological activity of EPN depends on numerous environmental factors. For example, 10–25 °C ,27,28. A10–25 °C ,30,31. N10–25 °C . The mot10–25 °C ,32.EPN populations are also influenced by biotic factors. The soil-dwelling infective juveniles are exposed to contacts with many organisms that share the same biotope, including bacteria, fungi and other nematodes. These organisms may reduce the EPN populations by food and territorial competency, or by their negative impact on EPN survival ,34.EPN are reported to be safe biological plant protection agents ,36, and The broad host range of most EPN and their potential to reproduce and survive in the soil environment are the main advantages for the use of EPN for biological pest control. Research data suggest that selection of native isolates of nematodes offers more benefits than using commercial preparations that are based on one specific nematode isolate, which is not necessarily local to the agroecosystem in question . StudiesIt is hypothesized that the activities of the isolates will be different, owing to their different origins and adaptations. It has been demonstrated that local populations of EPN can be more effective at reducing pests than the isolates recovered from other environments . StudyinS. feltiae with the use of morphometric and molecular methods, and by comparing the biological activities of isolated nematodes with the commercial EPN biopesticide tested on larvae of the greater wax moth .The aim of the work was achieved by identifying new environmental Polish isolates of Galleria mellonella for bioassays were obtained from laboratory cultures held at the Department of Microbiology and Parasitology, Jan Kochanowski University, Poland. Insects were cultured at 25 °C on beeswax patches in ventilated polypropylene containers. The fourth-instar of G. mellonella larvae, with an average body weight of 140 mg, was used for all analyses.2, using an Egner’s soil sampler. Ten organic farms on which wheat was grown were selected for the study . For EPNG. mellonella) [Under laboratory conditions, soil samples were mixed to obtain homogeneity and placed into 6 sterile 250 mL vessels. Field nematodes were isolated from soil samples using the insect trap method (lonella) . The samlonella) ) and cheDead moth larvae, cleaned of soil particles and washed three times with distilled water, were transferred to migration sponges (modified White traps—patent No. PL 212617 B1 ), and frG. mellonella were infected with 50 EPN juveniles/insect on Petri dishes with filter paper, which were then stored in the incubation cabinet at 20 °C over a 5-day period. The cadavers of insects parasitized by nematodes were transferred into migration Petri dishes [2 , and stored at 4 °C. Field EPN isolates were subjected to morphometric and genetic analyses for species identification.Larvae of i dishes  . The internal transcribed spacer (ITS) region was amplified by PCR using forward and reverse primers SF18SL (5′GTACACACCGCCCGTCGCTGC3′) and SF18SR (5′AAATCCTAGTTAGTTTCTTTTCCTCCGC3′) . ® Terminator v1.1 . The PCR reaction was performed in a Gene Amp PCR System 9700 thermal cycler . Purification of the reaction products was performed using the BigDye XTerminator Purification Kit . Reaction conditions were set according to the manufacturer’s instructions. The reaction products were separated using a 3130xl Genetic Analyzer capillary sequencer . Sample analysis was performed using the DNA Baser tool for the DNA Sequence Assembler program .The primers were used to determine the species. The PCR conditions were: 94 °C for 3 min and then 35 cycles , followed by an extension step at 72 °C for 5 min. After the last step, temperature was lowered to 4 °C, where the products were maintained until the purification step. The purification of the PCR product was performed using the PCR/DNA Clean-Up kit  and sequenced in the laboratory CoreLab of the Medical University of Łódź, Poland. The purified PCR products were sequenced using the appropriate primers SF18SL and SF18SR. PCR sequencing was carried out in a 10 µL reaction volume, using Big DyeS. feltiae species using BLAST, available on the NCBI website . The evolutionary relationship of the three isolates was inferred using the Neighbor-Joining (NJ) method with the phylogeny.fr  online software version 2021 for phylogenetic analysis [The ITS gene sequence of isolates iso1Lon (from Łoniów), iso1Dan (Danków Duży) and iso1Obl (Oblasy), obtained in this study, were compared to the GenBank nucleotide sequences of other analysis . S. feltiae isolate from the commercial Owinema biopesticide  was used as a control to compare the biological activities of the three field isolates . The insect mortality (percentage of insects killed due to any cause), infectivity (percentage of insects killed by nematodes), number of nematodes invading per insect, time to kill (days), time to first emergence of infective juveniles from infection (days) and number of infective juveniles emerging/insect were determined.The G. mellonella were infected with 50 EPN juveniles/insect on Petri dishes with filter paper (50 IJs/0.1 mL), which were then stored in the incubation cabinet at 20 °C. For each sample (three field isolates and control sample), 60 larvae insects were used.Larvae of S. feltiae isolates and one control isolate) were dissected in Petri dishes. Using preparation needles, the body of one dead larvae was dissected into small sections and the number of nematodes inside the insect body was counted under a light microscope (number of nematodes invading per insect).EPN infectivity was determined by dissection of insects 3 days after their death. Thirty insects from each study group  were transferred on migration sponges, and after observing the emergence of the first nematode juveniles, the nematodes were collected. Five harvests (one every two days) were performed over a period of nine days. The experiment was repeated twice.p < 0.05 used to differentiate homogeneous groups in multiple pairwise comparisons. Mean and standard deviation were calculated for the number of nematode juveniles migrating from one insect at successive harvests at two-day intervals and for the total number of migrating nematode juveniles from host insects from the different S. feltiae isolates.The Statistica version 13.3 software (statsoft.pl) was used for statistical analysis of data: morphometry and parameters of biological activity of nematodes. One-way analysis of variance (ANOVA) was performed for morphometric variables and the number of migrating infectious larvae. Mean and standard deviation were calculated for each morphometric variable in a given group. For biological activity parameters, Tukey’s test was used, with Steinernema feltiae was isolated from only three (Danków Duży (isolate iso1Dan), Łoniów (iso1Lon) and Oblasy (iso1Obl)) out of the ten regions sampled. S. feltiae was extracted from soils within a pH range of 5.2–5.4. The pH of the remaining soil samples was within the wider range of 5.2–5.6  .It was found that the number of nematodes invading per insect was significantly higher in the case of the iso1Lon isolate than with the other isolates .By analyzing the rate of development, it was shown that the control (Owinema) isolate and the iso1Dan isolate developed faster than the iso1Lon and iso1Obl isolates. A similar relationship was noted in the case of nematode reproduction, as the nematodes from the commercial preparation and the iso1Dan isolate started migrating within ten days of host infection, whereas the first progeny of the iso1Lon and iso1Obl isolates were observed migrating a day later .When examining the number of migrating infective juveniles on successive days of migration, was observed that the emergence of infective juveniles from insect cadavers with field isolates reached a peak on the fourth day of emergence, whereas the nematodes from the control sample reached a peak on the third day of emergence .p < 0.05) higher, in the range of 5000, for all three environmental nematode isolates than for the control sample  differences of invasion intensity and nematode reproduction between the field isolates of nematodes  and the control group  formed a subcluster with other The biological activity of the nematode isolates studied merits attention as it makes it possible to use local EPN isolates for the biological control of plant pests. The higher rate of survival and invasiveness by these field isolates, compared with the commercial biopesticidal isolate, would increase the quality and effectiveness of the biopreparation which could be prepared from one or more of these isolates.Recent studies have provided evidence that EPN isolates originating from various sources differ in their biological activity ,43,44,64EPN vary in their development rate ,65 and mIn this study, in terms of nematode biological activity (invasion extensiveness and intensity), the results were approximate to the results found by other authors from other countries ,67. The Other parameters of EPN bioactivity include their reproductive potential and their capacity to migrate from the host body. In the present study, it was demonstrated that nematodes isolated from the field needed more time to start migrating than did nematodes derived from the commercial biopesticide. A comparison of nematode reproductivity throughout the early days of migration showed that nematode isolates recovered from the field had a higher reproduction rate than did nematodes from the commercial biopesticide. Similar findings have been reported for these organisms in other studies ,65,68,69S. feltiae nematodes were isolated from sandy soil, one of the dominant agricultural soil types in Poland, as well as throughout northwest Europe . Therefore, the production of biopesticides from field S. feltiae isolates could have a wide economic impact for the protection of crops in Poland, as well as in other European countries.The field S. feltiae isolates originating from the Owinema biopreparation.The results obtained from this research confirm that field isolates are characterized by greater biological activity (higher invasion intensity and greater number of juvenile offspring from one host insect) than S. feltiae isolates for the production of effective biopreparations for crop protection.Additionally, this study supports the potential use of field entomopathogenic Steinernema feltiae was reported in cultivated soils in Poland. New isolates showed close genetic and morphological similarity to other S. feltiae isolates. The field nematode isolates were characterized by a similar insecticidal effectiveness, which was confirmed by comparable infectivity. Field nematode isolates exhibited a high reproduction rate and number of nematodes invading per insect capacity compared with S. feltiae from commercial biopesticide.


The aim of this study was to evaluate the influence of antirheumatic drug treatment on knee function of stiff knee patients after total knee arthroplasty (TKA).Twenty-seven patients (44 knees) of active RA (rheumatoid arthritis) or AS (ankylosing spondylitis) with stiff knees were included in this study. And they were divided into two groups according to continue antirheumatic drug treatment or not after TKA: the therapeutic group  and the controlled group . The outcomes were assessed by Knee Society Score (KSS), Visual Analogue Scale (VAS), range of motion (ROM) , “Forgotten Joint” Scale (FJS), with or without crutch, satisfaction, and revision (at year 2). The knee prosthetic loosening was evaluated by the followed X-ray at each following time.p < 0.05); however, the functional scores of KSS at month 6, year 1, and year 2 in the controlled group were more points improved. The therapeutic patients preferred the knee more at month 6, year 1, and year 2. The differences of KSS clinical scores , VAS, ROM, Crutch and FJS between the two groups were not statistically significant (p > 0.05).The mean follow-up time was 51 months (34–69 months). The KSS was higher at week 6 after TKA in the therapeutic group (For patients with stiff knees, the sequential antirheumatic drug treatment after TKA had no obvious effect on postoperative KSS, but can improve the satisfaction.Therapeutic level II. See Instructions for Authors for a complete description of levels of evidence. The amount of flexion achieved after total knee arthroplasty (TKA) is determined by the amount of preoperative flexion, especially if the flexion was less than 50° . TherefoStiffness can be defined as limited range of motion (ROM) of 50° or less that affects a patient’s ability to perform activities of daily living. One study found patients require an average of 83° knee flexion to climb stairs foot over foot. To sit in a chair without using one’s hands requires 93° knee flexion on average, and tying one’s shoes while seated requires 106° flexion on average [There were some studies regarding results of primary TKAs in patients with a preoperative arc of motion of 50° or less. From these literatures, we knew that the surgical approach in these patients could be a challenge because of difficulty in patellar eversion. Technical problems include lack of adequate exposure, the need for extensile surgical approaches, the risk of patellar tendon avulsion, difficulty balancing the flexion–extension gaps, component malpositioning, extensor mechanism management, patellar maltracking, avulsion of the collateral ligaments, and difficulty in wound closure . HoweverWe asked whether the sequential antirheumatic drug after TKA influences knee function, ROM, and satisfaction for stiff knee patients.In clinical work, it was found that some patients with RA or AS after TKA had spontaneously stopped rheumatology drug treatment for economic reasons or autonomous activity. We collected 30 patients with stiff knees and started follow-up observation from week 6 after TKA, when the control group stopped all postoperative and antirheumatic drug treatment.The inclusion criteria were as follows: (1) aged between 18 and 65, (2) all surgical procedures were conducted by the same surgeon, (3) all patients had a preoperative flexion arc of 50° or less in knees, and (4) a minimum follow-up period of 2 years. The exclusion criteria were as follows: (1) a history of septic arthritis of the knee or osteomyelitis, (2) any medical disability that limited the ability to walk and would not be considered suitable for a minimum 2-year follow-up period, (3) disabling diseases involving other joints of the lower extremities and severe deformities , (4) mental diseases, and (5) patients participating in other trials [These TKAs were performed by a senior author from January 2015 to October 2017. The patients were classified into two groups: Group 1 included stiff knees with sequential drug treatment after TKA , and Group 2 included stiff knees without sequential drug treatment   Tables , 4 and 5Radiographic evaluations of alignment were performed on preoperative and follow-up postoperative radiographs, which included anteroposterior (AP) standing, lateral and skyline patellar views, and full length of lower limbs. The data was evaluated by another independent orthopedic surgeon who did not know the patient’s sequential drug treatment. Data regarding the intraoperative and immediate postoperative complications were retrieved from the operating notes. Revision for any reason was documented.All patients received the total knee arthroplasty . The surgeries were performed by one senior physician under tourniquet control using a medial parapatellar approach. The posterior cruciate ligament was sacrificed because of severe deformity. We used an extramedullary alignment jig for the tibia and an intramedullary alignment jig for the femur. The patella was replaced in 11 knees . We performed a patelloplasty, which included soft tissue release from the lateral patella, division of the patellofemoral ligament, and patellar rim cautery to provide partial denervation and osteophyte removal. We removed 2–4 mm of the articular surface of the patella in TKA [We performed an extensile exposure (rectus snip) in the knees to enable eversion of the patella and knee flexion. Soft tissue procedures included lateral retinacular release and lateral gutter debridement . ExtensiPatients received antibiotic prophylaxis with intravenous ceftriaxone sodium . If the operation time exceeds 3 h or the blood loss is greater than 1500 ml, a second dose can be given during the operation, but no antithrombotic prophylaxis . The postoperative regimen included intravenous and oral analgesia , knee extension training immediately, gravity-assisted regaining of flexion, and walking with support from the second postoperative day. Progressive resistance exercises were started at day 2 postoperatively to strengthen the quadriceps and continued for 1 year postoperatively. All patients used walkers in 6 weeks postoperatively. The main drugs used in the sequential drug treatment group after TKA are methotrexate, sulfasalazine, tripterygium wilfordii, steroid, leflunomide, nonsteroidal anti-inflammation drugs, etanercept, and root of herbaceous peony (Table p < 0.05. Independent sample t tests were performed to determine the difference in age and body mass index (BMI). We used the Mann–Whitney test of nonparametric test for continuous variables when the data did not conform to the normal distribution, like the duration of hospital stay, operation time, blood loss, KSS, VAS, ROM, and FJS. Chi-squared test or Fisher's exact probability test were for categorical variables, like satisfaction and Crutch.SPSS 24.0 (SPSS Inc) was used for statistical analysis by an independent orthopedic surgeon. Clinical data were analyzed using means ± standard deviation or the median with the range in parentheses. The level of statistical significance was defined as We observed no differences in revision rates between the two groups Table . In group < 0.05) (Table p < 0.05) than that in patients without sequential drug treatment (group 2) at month 6, year 1, and year 2   Table . Howeverp = 0.435) (Table p > 0.05)  in group 1 and 25°  in group 2, without statistical significance p = 0.35 (Tablep = 0.335) (Table p > 0.05)  in group 1 and 6  in group 2, without statistical significance (5) Table . Postopep = 0.335).FJS scores at year 2 were 50  in group 1 and 75  in group 2 Table . The difp = 0.231)   Nonsteroidal anti-inflammation drugs; (2) glucocorticoids, local injection to treat refractory peripheral arthritis; (3) rheumatology medication, the drugs recommended by the American Academy of Rheumatology classification standards mainly include sulfasalazine and methotrexate and leflunomide, etc.; (4) biological agents, TNF antagonists are the first choice, which can improve the pain and function of the axial joints; (5) thalidomide, for special patients  .Due to severe pain, restricted mobility, and deformities in patients with advanced RA and AS, medical treatment can no longer meet the needs of patients; therefore, TKA is the best choice  as patiep > 0.05).Our series  was the first series of TKAs in stiff patients with limited ROM of 50° or less to verify this question. Our data observed that stiff knee patients with sequential antirheumatic drug treatment after TKA had a better clinical and functional outcome at week 6 after TKA than patients without antirheumatic drug treatment, but had lower scores in the functional part of KSS at month 6, year 1, and year 2. The clinical scores of KSS at other follow-up time were not statistically significant  activity level would increase in order to recover quickly, which made them have higher KSS functional scores at month 6, year 1, and year 2 after TKA. This situation was speculated to a certain extent that the KSS after TKA is related to the amount of activity.There was not much published information regarding results of TKAs in knees with a preoperative arc of motion less than 50°. Aglietti and Buzzi  reported ankylosed knees achieved less motion than stiff knees . MontgomPostoperative arc of flexion ranging from 64 to 103° had been reported in stiff and ankylosed knees in series ranging from 3 to 84 knees . Our resp > 0.05)  Table , but thep > 0.05)  Table . But thep = 0.335). This may indicate that patients with sequential antirheumatic drug treatment after TKA did not make sense to forget joint replacement.FJS scores at year 2 were 50  in group 1 and 75  in group 2 Table   after TKA would help relieve their general discomfort and control local inflammation Table .In addition, small bone sizes, severe osteopenia in a high proportion of patients with RA and AS, and severe soft tissue contractures made the operation technically demanding . During A constrained total knee prosthesis has been recommended for converting a fused knee to a TKA to substitute for deficient or absent collateral ligaments . In our McAuley et al. evaluated 27 TKAs in patients with a preoperative range of flexion less than 50° . They reOur study has several limitations. The primary limitation is that the study lacked adequate power to compare the results of TKAs in patients with knees ankylosed in extension with the results in patients with knees ankylosed in flexion. The other limitation is that several prostheses were used during the study, and the comparison of results based on the implant used also would be prone to inadequate power. To sit in a chair without using one’s hands requires 93° knee flexion on average, and tying one’s shoes while seated requires 106° flexion on average; more cases are needed for subgroup analysis from the two angles .Our results were inferior to results of a standard primary TKA and had a lower KSS and FJS . The surIn conclusion, for patients with stiff knees, the sequential antirheumatic drug treatment after TKA had no obvious effect on postoperative KSS, but can improve the satisfaction. And according to the result of a higher postoperative functional values of KSS in patients without sequential drug treatment, we considered more postoperative activity or better active awareness can improve postoperative function. We recommend patients with RA or AS undergo more activity in time after TKA.For patients with stiff knees, the sequential antirheumatic drug treatment after TKA had no obvious effect on postoperative KSS, but can improve the satisfaction.


Scirpophaga incertulas , and the striped stemborer (SSB), Chilo suppressalis , damage rice in Asia. YSB is the dominant species in much of tropical Asia. Both species are oligophagous on domesticated and wild rice. We investigated the roles of host plant preferences and larval performance in determining the larval densities of both species in rice plots. In screenhouse experiments, YSB showed significant preference–performance coupling. Adults preferred high-tillering rice varieties during early vegetative growth. In contrast, SSB did not demonstrate oviposition preferences under the same screenhouse conditions, but did oviposit less on the wild rice Oryza rufipogon Griff. than on domesticated rice varieties during a choice experiment. Despite differences in preference–performance coupling, larval survival and biomass across 10 varieties were correlated between the two species. YSB and SSB larvae occurred in relatively high numbers on rice varieties with large tillers  in wet and dry season field experiments. However, whereas YSB was the dominant species on IR68 and IR70, it was relatively less abundant on T16, where SSB dominated. Results suggest that YSB preferentially attacked fast-growing rice varieties with high tiller numbers early in the crop cycle. Meanwhile SSB, which has weak preference–performance coupling, occurred in rice plants with large tillers that were relatively free of YSB later in the crop cycle. These factors may allow the species to coexist. We discuss the implications of proximate and ultimate factors influencing stemborer co-occurrence for the sustainable production of rice in tropical Asia.Two stem-boring moths, the yellow stemborer (YSB)  Scirpophaga incertulas  [YSB]), the striped stemborer  [SSB]), the pink stemborer  [PSB]), and the white stemborer  [WSB])  . These s) [WSB]) , YSB is ) [WSB]) , and SSB) [WSB]) .Large-scale changes in rice production practices can cause shifts in the compositions of regional stemborer assemblages, including changes in species dominance. For example, SSB replaced YSB as the dominant species in southern Japan during the 1950s , and WSBMaliarpha separatella Ragonot: : : ; SSB: (Sricius): . Howeverricius): .2) or plot scales (100s of m2) under the influence of interspecific stemborer interactions.Strong preference–performance coupling will ensure that a species finds the most suitable hosts for its offspring. However, strong preferences may also have associated trade-offs where several species occupy similar niches. For example, because stemborer species are expected to select their preferred hosts based on the same host anatomical and nutritional requirements for developing larvae, then strong, shared preferences among coexisting species may be expected to promote interspecific aggregation and consequent competition. In contrast, differences in host preferences, or in the relative strengths of preference–performance coupling between species might promote coexistence. As a corollary, strong preference–performance coupling might be associated with a greater competitive ability, and weak coupling with a greater ability to disperse or otherwise avoid competition  of each species may occur . Both spOryza rufipogon Griff.; we included O. rufipogon because of a growing interest in developing resistant rice varieties through interspecific hybridization between domesticated rice and this species  Experimental Field Station at Los Baños, Philippines. Moths were collected using entomological nets during late evenings on the day prior to use in oviposition experiments. To acquire larvae for experiments, the egg masses were collected from the oviposition experiments (see below), measured using calipers, and maintained in individual glass vials until neonates emerged. The numbers of neonates emerging from each egg mass were recorded and the masses examined for unhatched eggs. Neonates were used in experiments within 1 h of egg hatch.O. rufipogon. Because of restrictions on experiments with wild rice species in open fields in the Philippines, we only conducted experiments with the second group of rice lines under controlled, screenhouse conditions. Seed was acquired through the IRRI Germplasm Bank with IR varieties acquired through the Plant Breeding, Genetics and Biotechnology Division of IRRI. For screenhouse studies, the seed was initially sown to seed boxes with fine soil and transplanted to paddy soil after 9 d. For field experiments, the seed was sown to raised seedbeds in a screenhouse and transplanted to field plots after 28 d.We used 10 rice varieties in our main experiments. The rice varieties TKM6, Taitung 16 (T16), IR36, IR40, IR50, IR62, IR66, IR68, IR70, and IR72 were selected based on unpublished records from IRRI of varietal screening for resistance development to stemborers . The screening tests indicated a range of susceptibilities to stemborers among the varieties. In a second group of experiments, we used the varieties IR62 and IR64, and the wild rice species O. rufipogon). The experiments were conducted as follows.Oviposition choice experiments were conducted in a screenhouse facility. The screenhouse consisted of concrete-walled, mud-filled trenches that simulate field conditions, but without much of the insect and disease pressures of field conditions because the house was clad with 1 mm insect netting. We conducted three separate oviposition experiments. In the first two experiments, SSB (experiment 1) or YSB (experiment 2) females were allowed to freely oviposit on 10 rice varieties . In experiment 3, females of SSB and YSB (in separate cages) were allowed to freely oviposit on three rice lines  of each variety were transplanted at one seedling per hill to microplots in the screenhouse facility. In experiments 1 and 2, each microplot consisted of 30 hills (10 varieties × 3 hills) distributed as 5 rows with a spacing of 35 × 35 cm between hills. The varieties were randomized within each microplot. The microplots were individually covered with mesh cages of dimensions 2.45 × 2.10 × 1.4 m (L × W × H) replicated six times. In experiment 3, microplots consisted of one plant of each of the three test lines planted side-by-side with 25 cm spacing. Plants were randomized among the three positions and the plots were covered with mesh cages of dimensions 1.0 × 0.5 × 1.4 m (L × W × H). The entire experiment consisted of 12 cages (2 moth species × 6 replicates).Fertilizer was applied to plots in all experiments at a rate of 100 kg of nitrogen/hectare 10 d after transplanting (DAT). At 20 DAT, the cages were each infested with 25 (experiments 1 and 2) or 2 (experiment 3) gravid females of either YSB or SSB. After 5 d, the cages and adults were removed and the plants were searched for egg masses. The number of egg masses per hill was noted and the masses collected and individually stored in scintillation vials to allow larvae to emerge. The numbers of larvae emerging from each egg mass were recorded.We assessed stemborer performance in the same microplots that were used in the choice oviposition experiments (see above). The protocol for each of the three experiments was the same. At 35 DAT (mid-tillering), when all egg masses had been removed from the microplots, each plant was infested with 10 neonate YSB or SSB using a fine paintbrush. Neonates were carefully placed on individual tillers (one per tiller), above the water line. The delay in infesting, after removal of all egg masses, was determined by the time for neonates to emerge from the egg masses (as observed in the laboratory). After infestation, the larvae were allowed to develop on the plants for 35 d after which the plants were destructively sampled. Plants were pulled from the soil and the numbers of tillers, including dead tillers (dead heart), were recorded. The stems were carefully examined for larval entry holes and were dissected to collect developing larvae and pupae. The larvae and pupae were counted, dried, and weighed. Survival was calculated as the proportion of the original 10 neonates per plant that were found alive. Development was measured as the proportion of larvae than had developed to the pupal stage at the time of sampling. The plants were placed in paper bags and dried in a forced draught oven at 60°C for 2 wk before being weighed.2 with different rice varieties. Plots were flooded until pre-harvest and received fertilizer (ammonium sulphate) equivalent to 100 kg (wet season) or 150 kg (dry season) of nitrogen per hectare as a basal treatment, at 3 wk after transplanting, and again at panicle initiation. Solophos, muriate of potash and zinc were applied basally with the ammonium sulphate. Plots received no pesticide treatments. Seed was initially sown to screen house seedbeds and 28-d old seedlings transplanted as one plant per hill to the puddled field plots. Hills were spaced at 25 × 25 cm (planting distance). Further details of each experiment are presented below.We conducted two field experiments with natural stemborer infestations to examine preferences and performance of stemborers in field plots with different test varieties. In both of the field trials, we focused on early crop stages (≥60 DAT – seedling to booting). The experiments were conducted during the wet and dry season at the Experimental Field Station of IRRI. The field site at IRRI had deep clay soils with about 4% organic matter. The experiments each consisted of field plots of <104 mDuring the wet season, field plots of 5 × 5 m were planted with seedlings of one of each of the 10 rice varieties that were used in oviposition choice experiments 1 and 2 described above . The plots were planted as six replicated blocks (completely randomized block design) with five rice hills of TN1 between adjacent blocks.The rice plots were examined at 15 DAT (early tillering) for stemborer egg masses by carefully examining each hill. Rice plots were randomly sampled for pest and disease incidence at 30 DAT (mid-tillering) and 60 DAT (booting)  and weighed . After examination in the laboratory, the plants were separated into different parts  and dried at 60°C in a forced draught oven until a stable weight.During the dry season field experiment, 5 blocks, each with 4 plots of 8.25 × 12.5 m (L×W) were planted with IR66, IR68, IR70, and IR74  in a randomized block design. The varieties were selected on the basis of relatively low (IR66 and IR72) and high (IR70 and IR68) stemborer damage  in the previous wet season field experiment. The plots were examined at 15 DAT (early tillering) for stemborer egg masses. Individual plants were randomly sampled from each plot for stemborer incidence at 30 DAT (mid-tillering) and 60 DAT (booting), and the plants were processed in the laboratory as described above. No control, stemborer-free rice plants were selected from plots during the dry season experiment.The relations between egg mass length and the numbers of eggs per mass were analyzed using linear regression. Results from screenhouse choice oviposition experiments  were analyzed separately for each stemborer species using univariate general linear models (GLM) with variety as the main factor and removing the effects of blocks. Data were ranked because of nonindependence of measures. Stemborer performance on the rice lines in the screenhouse was analyzed using GLM with one (variety) factor for damage, survival, total insect biomass and development times or two factors  for pupal weights, and removing the effects of blocks. Tiller number was initially included in the analysis as a covariate, but was removed where there was no significant effect. We conducted a range of Spearman’s correlations between the results from the screenhouse oviposition and larval performance experiments to assess preference–performance relations for each stemborer species. Spearman’s correlations were also used to examine relations between oviposition and performance of the two stemborer species and to assess the relations between tiller number and plant weight on stemborer fitness (preference and performance). Results (stemborer numbers) from the field trials were analyzed using multivariate analysis of variance (MANOVA) that included YSB, SSB, and PSB larval numbers as dependent variables. Variety was the main factor examined and block effects were removed. MANOVAs were conducted separately for each sampling date. Tukey’s HSD tests were conducted for all significant GLM and MANOVA results. Following analyses, residuals from each parametric test were plotted and assessed for normality and homogeneity. Data transformations are indicated with statistical results. Multiple regression with backward elimination was used to determine the best predictors of field infestations during the wet season experiment. We used data from the noninfested plants  as independent variables and larval numbers as dependent variables. We also included the combined numbers of heterospecific stemborer larvae, whorl maggot damage, and damage from other lepidopterans as independent variables at the initiation of each analysis. Residuals were plotted following the analyses to ensure normality and homogeneity of variances.F1,60 = 48.416, P < 0.001; covariate length F1,60 = 95.959, P < 0.001). Egg mass length was related to egg number in both species with a greater slope for YSB  or SSB  egg masses on either IR62, IR64, or O. rufipogon in experiments with three rice lines ; however, fewer larvae emerged from SSB egg masses on O. rufipogon  on the varieties   . Only thO. rufipogon compared to IR62 or IR64 . Survival  and female pupal weights  of YSB and SSB across varieties, and the damage caused by each species to the rice varieties  were not correlated.There were no significant correlations between egg laying by YSB and SSB or the number of larvae of each species emerging from egg masses collected on the 10 rice varieties (egg masses: Xanthomonas oryzae) . There was no significant effect of rice variety on the numbers of larval YSB , PSB  or SSB  occurring in tillers during early wet season sampling . Significantly more SSB larvae occurred on T16 than on any of the remaining varieties, except IR68 , but there was no effect of variety on the numbers of PSB larvae occurring on the plants , average tiller weight (P = 0.032), and the number of leaves per tiller (P = 0.003) were the best predictors of YSB numbers during early sampling . Shoot weight (P = 0.009) and leaf weight (P = 0.031) were the best predictors of YSB numbers during the later sampling . There were no statistically significant predictors of SSB numbers across varieties during the same sampling.The number of tillers . More YSB larvae  and PSB larvae  occurred on IR68 than on the other three varieties during early sampling. SSB larvae were only detected on IR70 at that time , and SSB  occurred on IR68 compared to the other varieties, and more PSB occurred on IR68 compared to IR72 . In particular, the high numbers of SSB on IR68 and T16 in the wet season field experiment contrasted with relatively low preferences for these varieties by the same species in the screenhouse study. There was also no significant correlation between egg laying by moths and larval numbers on varieties in the wet season experiment.Field densities of YSB and SSB eggs and larvae from the wet and dry season experiments were not correlated with screenhouse preferences or any performance parameters from the screenhouse study  and in the present study, fewer SSB larvae emerged from eggs laid on O. rufipogon than on two O. sativa ssp indica varieties from a choice experiment. Although performance was similar on these rice lines in our experiments, previous experiments have indicated SSB to perform poorly on O. rufipogon as the wild rice species matures   . This suThe larvae of SSB, although not gregarious, often occur in larger numbers on individual rice tillers compared to YSB. In our wet and dry season field experiments, 98.8% of YSB-infested tillers, 98.4% of PSB-infested tillers, and 94.0% of SSB-infested tillers contained only a single larva. Larvae of all three stemborer species were present together in a small number of plants—but no tiller contained more than a single species. For SSB, a single tiller could contain up to five larvae. Little is known of the relative dispersal abilities of YSB or SSB, however, -1, reproductive stage = 17–20 plant-1), whereas T16 produces a small number of tillers that gradually gain weight without significant loss of tillers after maximum tillering  compared to the screenhouse (50 DAT—maximum tillering). This was possibly accelerated by the relatively higher densities of plants in the field trials and the greater intensity of light under field conditions. Meanwhile, IR68, IR70, and T16 had relatively high stemborer densities in the field experiments compared to the other varieties. This again contrasted with the screenhouse results, which indicated IR70 and T16 as among the least suitable hosts for both of the stemborer species. Multiple regression analysis using field data suggested that shoot and tiller weight are among the best predictors of YSB densities: both IR68 and T16 produce heavy tillers and were among the tallest plants from among the varieties exposed in the wet season field experiments . There are several examples of herbivores avoiding plants previously attacked by potential competitors. Evidence suggests that changes in plant volatiles emitted from damaged plants can alter the behavior of subsequent herbivore colonizers . AdditioRice breeders, interested in developing rice varieties with resistance to stemborers have generally focused on single species from target stemborer assemblages (74% of 207 published papers: Horgan unpublished). SSB has been the predominant species studied in rice breeding programs in recent years. Results from comparative studies of SSB and YSB, including the present study, suggest that whereas larval performance in both species, or plant responses to attack by both species may be correlated across varieties, preferences are not . Therefonvab034_suppl_Supplementary_InformationClick here for additional data file.


Notably, both estimates of hyperalgesia were significantly correlated . Comprehensive somatosensory testing (DFNS standard) revealed that no test parameter was changed significantly following electrical HFS. The experiments demonstrated that fascia stimulation at a sufficient stimulus intensity elicited significant across-tissue facilitation to pinprick stimulation , a hallmark sign of nociceptive central sensitization.Musculoskeletal pain is often associated with pain referred to adjacent areas or skin. So far, no study has analyzed the somatosensory changes of the skin after the stimulation of different underlying fasciae. The current study aimed to investigate heterotopic somatosensory crosstalk between deep tissue (muscle or fascia) and superficial tissue (skin) using two established models of deep tissue pain   or the injection of hypertonic saline in stimulus locations as verified using ultrasound). In a methodological pilot experiment in the TLF, different injection volumes of hypertonic saline (50–800 µL) revealed that small injection volumes were most suitable, as they elicited sufficient pain but avoided the complication of the numbing pinprick sensitivity encountered after the injection of a very large volume (800 µL), particularly following muscle injections. The testing of fascia at different body sites revealed that 100 µL of hypertonic saline in the temporal fascia and TLF elicited significant pinprick hyperalgesia in the overlying skin (–26.2% and –23.5% adjusted threshold reduction,  The most prevalent musculoskeletal pain conditions transitioning to chronicity and contributing to the global burden of disease include low back pain, neck pain and temporomandibular disorder pain ,2. In maWe hypothesized that painful chemical stimulation  of the muscle and fascia of the lower back will result in a stimulation intensity-dependent heterotopic sensitization of the overlying skin to punctate mechanical stimuli, a hallmark sign of nociceptive central sensitization . FurtherSomatosensory evocations and changes of the skin were attempted via the electrical and chemical stimulation of the underlying thoracolumbar fascia (TLF) and multifidus muscle (MM) of the lower back. Herein, the chemical stimulation was applied using several stimulation intensities. To investigate the differences in the central nociceptive processing input of fascia tissue, the chemical stimulation protocol was further applied at other body sites, namely the temporal fascia, trapezius fascia, and iliotibial band (IT band). Forty-eight young healthy volunteers  with no history of back pain participated in this study, sixteen each for the three different experiments. All volunteers signed a written consent form and had sufficient command of the German language. The criteria for exclusion were the use of any medication or having undergone recent surgeries. None of the participants withdrew from the study prematurely. Local ethics committee approval was obtained according to the current version of the Declaration of Helsinki . 2, CareFusion, USA) were positioned under ultrasound guidance  bilaterally into the thoracolumbar fascia , and inp fascia ,11,20. HIn an exploratory dose-finding pilot experiment, three different amounts  of hypertonic saline (5.8%) were injected into the multifidus muscle and the overlying thoracolumbar fascia (deep fascia ) at spinIn 16 healthy volunteers , we injected 100 µL of hypertonic saline at different fascial sites to compare the impact of chemical stimulation in different fasciae. These injection sites were the iliotibial band (IT band) at a mid-distance between the greater trochanter and the fibular head at the lateral midline, the TLF approximately 2 cm lateral to the midline at the L3/L4 level overlying the multifidus muscle, and the fascia nuchae approximately 3–4 cm lateral to the midline at the C7/Th1 level overlying the trapezius muscle . In addThe comprehensive QST battery of the German Research Network on Neuropathic Pain (DFNS) ,42 was pA Peltier-based computerized thermal stimulator , with a 3.2 cm × 3.2 cm contact probe was used. Thermal thresholds were measured using ramped stimuli (1 °C/s) until the subject pressed a stop button signaling that they had reached the threshold for detection or pain (method of limits). All thermal ramps were started from 32 °C baseline temperatures. Cut-off temperatures for tissue safety were 0 °C and 50 °C. First, the cold and warm detection thresholds  were assessed. This was followed by a sequence of alternating warmth and cold stimuli, three of each. In this thermal sensory lime (TSL) procedure, subjects were asked to report the quality of each of the subsequent thermal stimuli. Mistaking one or more of the three cold stimuli during TSL as warm, hot, or burning pain was noted as the occurrence of paradoxical heat sensations (PHS). Afterwards, the cold and heat pain thresholds  were investigated. Here, pain indicates any “burning”, “stinging”, “drilling”, or “aching” sensation. Subjects were not permitted to look at the computer screen during thermal testing and were not given visual or auditory cues to indicate the start of the stimulus. Mechanical detection threshold (MDT) testing for A-beta fiber function (SAI slowly adapting mechanoreceptors) was assessed with a set of calibrated von Frey filaments  that exerted forces between 0.25 and 512 mN. Using an adaptive method of limits, five ascending and descending series of stimuli applying consecutive stimulation forces were applied, and the geometric mean was calculated for the final tactile threshold.The mechanical pain thresholds for pinprick stimuli (MPT) testing for A-delta fiber mediated hyper- or hypoalgesia to pinprick stimuli ,44 were In a separate test, a stimulus–response function for the mechanical pain sensitivity (MPS) was determined using the same pinpricks as described above. Following each stimulus, the subject was asked to rate the magnitude of pain on a verbal numerical rating scale . Pain to light touch  (which tests A-beta fiber mediated pain sensitivity to light touch) was tested via light stroking with a cotton wisp (3 mN), a cotton wool tip fixed to an elastic strip (100 mN), and a soft make-up brush (400 mN). Volunteers were asked to use the same NRS (see above). At every test site, each of the seven intensities of pinpricks and three intensities of light stroking were applied five times in a balanced order. The mechanical pain sensitivity was calculated as the geometric mean of all pain ratings for pinprick stimuli. The dynamic mechanical allodynia was quantified as the geometric mean of all numerical pain ratings after light touch stimuli. To avoid a loss of zero-values, a constant of 0.1 was added to each rating for both analyses, for mechanical pain sensitivity and for dynamic mechanical allodynia. For more details on the evaluation of MPS (see ).Assessment of the stimulus response function for pricking pain was followed by an assessment of pain summation tested by the wind-up ratio (WUR). WUR was examined using a train of 10 pinprick stimuli of 256 mN (repeated at 1 Hz) and comparing the painfulness of this train of stimuli to the pain elicited by a single pinprick stimulus of 256 mN. The wind-up ratio was calculated as the mean pain rating of five series of repetitive pinprick stimuli divided by the mean pain rating of five single stimuli.Vibration detection threshold (VDT) testing for A-beta fiber function (rapidly adapting mechanoreceptors) was examined as a flutter–vibration threshold using the semiquantitative 64 Hz Rydel–Seiffer tuning fork that has a graded readout of vibration amplitude (0–8/8 units). Vibration detection threshold was assessed with three series of descending stimulus intensities over spinous process of the lower back (L3). 2. The algometer was pressed on the skin with an increasing ramp of 0.5 kg/s and the subject was asked to respond verbally as soon as the pressure became painful. As in all mechanical pain testing procedures, pain was indicated by any “sharp”, “pricking”, or “stinging” sensation. Finally, the pressure pain thresholds (PPT)—which is the only test for deep pain sensitivity in this QST battery, most probably mediated by nociceptive muscle C- and A-delta fibers—were measured using a pressure algometer . The algometer had a rubber tip, with a contact area of 1 cmbaseline)/SDbaseline. This standardization returns dimensionless standard values of 0 ± 1 SD for the baseline data. Deviation from the baseline is thus shown in units of SD of the baseline. The sign of this transformation was chosen to give negative values for reduced sensitivity (loss) and positive values for increased sensitivity (gain). The same transformation was also used for MPT in different tissues. Statistical analysis was performed using SigmaPlot software; version 12.5  or Statistica, release 4.5 . Significant differences  were determined by repeated measure analysis of variance (RM-ANOVA), followed by a Fisher–LSD post hoc test. All values given in this study are depicted as mean ± SEM. QST values were analyzed as described previously ,42,43. TUnexpectedly, the mean pain intensity elicited by focal electrical HFS stimulation in muscle or fascia was only 10.6/100 NRS and 18.0/100 NRS, respectively (data from ). This wMoreover, subjects never reported the occurrence of thermal dysesthesia )) or mechanical dysesthesia ; data not shown)). CDT, cold detection threshold; CPT, cold pain threshold; HPT, heat pain threshold; MDT, mechanical detection threshold; MPS, mechanical pain sensitivity; MPT, mechanical pain threshold; PPT, deep pain sensitivity to blunt pressure; TSL, thermal sensory limen; VDT, vibration detection threshold; WDT, warm detection threshold; WUR, wind-up ratio.Assuming that the HFS of deep tissue was not sufficiently painful to induce heterotopic facilitation in the skin, we proceeded to the well-established model of chemical stimulation via hypertonic saline injection ,37,38,46Hypertonic saline injections elicited a dose-dependent pain lasting for several minutes. From the beginning, the smallest injection volume (50 µL) was about 50% more painful than HFS, namely 15.8 ± 13.9 NRS in the muscle and 25.1 ± 16.1 NRS in the fascia at peak pain (mean ± SD). The medium volume (200 µL) elicited a mean peak pain of 29.5 ± 15.7 and 45.0 ± 17.2 NRS, respectively. The largest volume (800 µL) elicited even stronger pain in both tissues, namely 35.6 ± 18.0 NRS following muscle injections and 53.9 ± 19.1 following fascia injections. In the most pain-sensitive subjects, peak pain ratings came close to or even exceeded the pain tolerance limit. Hence, pain ratings after chemical stimulations were approximately 1.5–3 times higher than pain ratings elicited by electrical HFS.p = 0.133) or “injected volume” , nor was there a statistically significant interaction between both .Moreover, there were no significant higher order interactions , while smaller injection volumes did not. Moreover, this numbness of the skin was more significantly pronounced after muscle injection compared to that following fascia injections , for the largest volume  in particular. Accordingly, we concluded that very large painful injection volumes likely obscured the potentiating effect of hypertonic saline, when injected into the muscle in particular.Four-way RM-ANOVA of mechanical pain thresholds (MPT) revealed that there were no robust differences between tissue types or injected volumes as a significant main effects of “tissue” , we stimulated the fascia in four different areas along the body axis  and lateral thigh , and the more proximal areas of the trapezius muscle fascia or TLF), eliciting pain intensities approximately two times higher in overall mean compared to the HFS of the TLF. Data on pricking pain thresholds (MPT) were analyzed via RM-ANOVA .3,45 = 4.48, p < 0.01). Three of the test areas  exhibited almost the same MPT of approximately 35 mN and differed by <1 mN in their average MPT . In contrast, the skin overlying the iliotibial band exhibited an MPT approximately 60% higher than that of any other test area  and a complex three-way interaction of “before vs. after”, “control vs. test sites”, and “test areas” . The complex interaction could be stepwise broken down into several differences. After injection, the injected sites exhibited an overall reduced sensitivity to pinprick . However, with a closer inspection, this was only significant in the control sites, with an average increase of MPT by +20.2% (p < 0.01 post hoc LSD test), but it was much weaker in the test sites chemically stimulated with hypertonic saline . Comparing the test areas individually revealed that the desensitization was significant at the control site in the temporal area  and the TLF area . In contrast, there was no significant reduction of sensitivity (MPT increase) following injections into the iliotibial or trapezius areas ; a similar drop of MPT was also encountered in the TLF area  and –23.5% (p < 0.05), respectively, relative to the control site. It also disclosed that local tenderness was only identified as a pain sensitivity increase when the general numbness was controlled by the assessment of MPT in unconditioned control sites , and MPT was even increased after hypertonic saline in the iliotibial band . Changes of MPT in these areas also did not correlate to those in the hyperalgesic areas . Accordingly, when successfully elicited, there was a remarkable intraindividual stability of the hyperalgesia expression and highly differentiated response patterns across the different test areas.Furthermore, in the same two areas, test sites injected with hypertonic saline were significantly more pain sensitive than their corresponding control sites after injection. In particular, the temporal test area was more pain sensitive than the control site (pain facilitation), with a pronounced relative drop of MPT . The elicitation of this across-tissue pain facilitation could not be easily demonstrated because it depended on the nature of the conditioning stimulus and of the stimulated deep tissue. Here, it could be elicited using a validated model of chemical stimulation  ,37,38,46Punctate hyperalgesia is the most prominent sensory sign of the central sensitization of nociceptive projection pathways ,24,25,52Notably, very shortly after Clifford Woolf—based on electrophysiological animal experiments—had first proposed that central sensitization contributed to hyperalgesia , furtherThe asymmetry of mutual facilitation between fascia and muscle can be explained by differences in their innervation. The fascia is much more densely innervated by peptidergic nociceptors, which are the decisive drivers of central sensitization ,63,64,65It has previously been shown that experimental and clinical pain associated with hyperalgesia was accompanied by reduced tactile sensitivity ,70. In pInterestingly, the pain inhibition was twice as strong when injections targeted the muscle compared to injections targeting the fascia. Together with a lesser capacity of the noxious muscle stimulation to induce hyperalgesia, this finding further supports the concept that the balance between induction of diffuse noxious pain-inhibiting controls (DNIC) suppressing pain sensitivity in a spatially nonselective fashion and pain-focal facilitating mechanisms differs between fascia and muscle, making the fascia a more likely source of hyperalgesia induction. Thus, affection of the muscle may be more prone to elicit numbness, while affection of the fascia leans towards an elicitation of tenderness.However, the induction of hyperalgesia following fascial injections could not to be easily generalized, since hyperalgesia was only induced by hypertonic saline injections of the temporal fascia or thoracolumbar fascia, but not in the trapezius region or iliotibial band. Different fasciae have widely different forms and functions ,78,79. TThis study has shown that the chemical stimulation of deep tissues (muscle and fasciae) can cause highly differentiated response pattern across the different tissues and test areas. The experiments demonstrated that fascia stimulation , at sufficient stimulus intensities, elicited significant across-tissue tenderness to pinprick (applied to the overlying skin), which is a hallmark sign of heterotopic nociceptive central sensitization. Nonetheless, very large painful injection volumes likely activated the endogenous pain control system, which obscured the potentiating effect of hypertonic saline. This inhibition was twice as strong when injections targeted the muscle, compared to injections into the fascia. Together with a lesser capacity of noxious muscle stimulation to induce hyperalgesia, this finding further supports the concept that the balance between pain-inhibiting and pain-facilitating mechanisms differs between fascia and muscle, making the fascia a more likely source of hyperalgesia induction.