Hearing loss is prevalent and disabling, yet little is known about the extent of recreational noise exposure and hearing protection use. The aim of the present research was to estimate the extent of recreational noise exposure and hearing protection use in a sample representative of the UK adult population.We conducted a cross-sectional survey of 10,401 UK adults who were representative of the population.n = 7590, 73.0%) reported exposure to recreational noise excluding headphone and earphone use in the last 12 months. Just 158 people (2.1%) reported wearing hearing protection for every noisy recreational activity. Age (younger people) and beliefs of a behavioral (as opposed to genetic) cause of hearing loss were predictive of both higher recreational noise exposure and greater hearing protection use. Men were more exposed to recreational noise but women were less likely to use hearing protection.More than 7000 people  causes of hearing loss. Hearing loss is ranked 4th globally in years lived with a disability, with older age and noise exposure being the biggest risk factors . ApproxiNumerous studies have tried to estimate recreational noise exposure and hearing protection uptake , but have typically been limited to convenience –13 or reWithout knowledge of the extent to which the general public are exposed to recreational noise and what is the prevalence of hearing protection device use, it will be difficult to develop interventions to reduce the health costs that might arise in the future nor what interventions might promote use of hearing protection. The aims of the present research are, for the first time in a representative sample of the UK adult population, to: (a) assess the extent of recreational noise exposure, and (b) assess hearing protection use.Once the frequency of recreational noise exposure and prevalence of hearing protection use is established, the question then arises as to what are the correlates of exposure and use that will shed light on the kinds of populations and constructs that may be targeted/changed in future interventions. We therefore additionally examined potential correlates of recreational noise exposure and hearing protection use, in addition to sociodemographic variables and clinical characteristics , we also considered beliefs about whether hearing loss is caused by genetics or health behaviors  to see wThe study design was cross-sectional and administered via an online survey.A sample of adults designed to be representative of the UK adult (18+ years) population was invited by YouGov, a market research company, to take part in an online questionnaire in March 2019. YouGov has a panel of over one million potential respondents from whom they successfully recruited a sample of 10,401 UK residents, based on a 90.6% completion rate. Participants were incentivized in accordance with YouGov’s points system, whereby respondents accumulate points for taking part in online surveys and the points can be traded for cash and gift cards. The data were sent securely to the research team for analysis. Ethical approval was obtained from a University of Manchester ethics committee (ref: 2019–5769-9246) and participants gave written informed consent by clicking on a series of boxes at the beginning of the online survey.A questionnaire, designed specifically for the purposes of the present study, was embedded in a wider online anonymous survey. We asked questions about participants’ sociodemographic and clinical characteristics, recreational noise exposure, hearing protection use and beliefs about the causes of hearing loss. These questions are described briefly in the section below and are presented in the Additional file Sociodemographic measures of gender, ethnicity and socioeconomic status were taken using standard UK Office for National Statistics 2016) measures. Consistent with the ethnic profile of the UK, ethnicity was divided into White versus Black, Asian or Minority Ethnic. We gathered data on twelve occupational categories , nightlife , making music , DIY , engine noise , sports related noise  or cinema.” Following this, participants were asked, “How often did you take part in noisy leisure activities during the last 12 months?” , 15 to wUse of hearing protection during exposure to noisy leisure activities was measured using the item, “How often, if at all, do you use earplugs and/or earmuffs to protect your hearing when you take part in noisy leisure activities?” to which participants responded on an eleven-point scale from 0 to 100% that had 10% intervals, which was adapted for questionnaire use from Lutman et al. .Data were weighted to ensure analyses properly reflected the UK population. Descriptive statistics were used to characterize the population. Linear multiple regression was used to predict recreational noise exposure and logistic regression was used to identify predictors of hearing protection use.SD = 1.48; 18–93 years). Most people (75.9%) did not report a hearing loss and did not have people in their family with hearing loss (67.4%). Approximately half (49.8%) of the sample believed that hearing loss was caused by genetics whereas just 16.8% thought that hearing loss had a behavioral cause.Consistent with the sampling frame, the sample was broadly representative of the UK population Table . Most pan = 6732) reported experiencing recreational noise less than once a month  reported exposure to excessive recreational noise on at least a weekly basis. Multiple regression  reported some exposure to recreational noise in the last 12 months. Of those participants reporting some recreational noise exposure, n = 6007 (79.2%) reported zero use of hearing protection during noisy recreational activities in the last 12 months  interventions to reduce exposure might best be targeted at men and younger people, and that more needs to be done in terms of explaining that there is a behavioral underpinning to hearing loss; and (b) interventions to increase hearing protection use might best be targeted at women and older people with challenges made to the belief that hearing loss has a genetic cause.Ensuring that people are aware of the behavioral underpinnings to hearing loss is something that could be implemented at a population level, rather than targeting specific groups and it is notable that exposure was associated with greater reported hearing loss. The implication is that perhaps exposure drives the belief that hearing loss has a behavioral as opposed to genetic cause of hearing loss, which chimes with research conducted in the US showing that the presence of hearing symptoms contributed to greater negative attitudes towards noise . The impAlthough the present research takes the literature on the prevalence of recreational noise exposure and health protection use forward in some important respects, it is important to note some potential limitations. First, although the sample was large and representative, the cross-sectional design of the study means that causality cannot be inferred. Second, the nature of the research meant that exposure and use of hearing protection were self-reported, rather than assessed objectively. It would be valuable in future research to harness the power of new technology to gauge population exposure to recreational noise and use of hearing protection unobtrusively with a greater level of accuracy. Third, it is worth highlighting that, as a function of our interest in both recreational noise exposure and hearing protection use, our definition of noisy recreational activities excludes headphone and earphone use. This means that our estimate of exposure is likely to be lower than what has traditionally been considered to be total recreational noise exposure .In sum, a significant proportion of the UK population are exposed annually to recreational noise, yet few take protective action. Younger people and men might be appropriate target groups to reduce exposure; whereas older people and women might be targeted with interventions to increase hearing protection use. However, a population-level approach to improving hearing health might usefully focus on ensuring that people have greater awareness of the behavioral causes of hearing loss.Additional file 1.


No significant differences in telocytes morphology and immunophenotype were found in patients with various forms of AF. CD68-positive cells were detected more often in AF + IAA than AF group. We assume that in aged AF + IAA patients remodeling of atrial myocardium provoked transformation of telocytes into “transitional forms” combining the morphological and immunohistochemical features with signs of fibroblast-, histiocyte- and endotheliocyte-like cells.Telocytes are interstitial cells with long, thin processes by which they contact each other and form a network in the interstitium. Myocardial remodeling of adult patients with different forms of atrial fibrillation (AF) occurs with an increase in fibrosis, age-related isolated atrial amyloidosis (IAA), cardiomyocyte hypertrophy and myolysis. This study aimed to determine the ultrastructural and immunohistochemical features of cardiac telocytes in patients with AF and AF + IAA. IAA associated with accumulation of atrial natriuretic factor was detected in 4.3–25% biopsies of left (LAA) and 21.7–41.7% of right (RAA) atrial appendage myocardium. Telocytes were identified at ultrastructural level more often in AF + IAA, than in AF group and correlated with AF duration and mitral valve regurgitation. Telocytes had ultrastructural signs of synthetic, proliferative, and phagocytic activity. Telocytes corresponded to CD117 TCs morphology is similar to Cajal pacemaker cells of the gastrointestinal tract. TCs are usually spindle-shaped and differ from all known types of connective tissue cells by the presence of thin processes , the length of which reaches 100 μm. In recent years, the possible role of cardiac TCs has been actively discussed. The processes of the TCs “accompany” all components of the interstitium—cardiomyocytes, blood vessels, nerve fibers. TCs contact with each other and with other interstitial cells due to nexuses, point contacts, or nanocontacts30. They are performed a structural and a coordination bond between all cell types32. TCs number in atrial myocardium is higher than in ventricles, and in epicardium is higher than in endocardium33. It is assumed that in atrial myocardium and in myocardial sleeves of human pulmonary veins, TCs have pacemaker activity2. They probably act as alternative generators of electrical impulses. TCs have direct nanocontacts with endotheliocytes and thus provide structural and functional support for endothelial cells, create a microenvironment, and participate in angiogenesis36. Moreover, TCs organize the architectonics of myocardial tissue and determine the localization and differentiation of stem cells in the processes of growth and regeneration of cardiac muscle tissue38, similar to those occurring in skeletal muscle during the interaction of TCs with resident satellite cells29.Telocytes (TCs)—is recently discovered interstitial cells described in the myocardium44. Amyloidosis is a process associated with impaired protein metabolism and is accompanied by the formation and deposition in tissues of a specific protein-polysaccharide complex—amyloid. Amyloidosis is usually a systemic disorder with multiple organ damage affected. Nevertheless, the myocardium of patients with AF is characterized by a heart-specific type of amyloidosis—IAA. According to some authors, IAA is associated with the deposition of a fibrillar form of atrial natriuretic peptide (ANP) in atrial myocardium50. Normally, ANP is predominantly synthesized by atrial myocardium, found in electron-dense atrial granules in cardiomyocytes, and released in response to atrial wall extension. ANP activates transmembrane receptors in target organs, has a natriuretic, diuretic, and vasodilatory effect, and inhibits the renin–angiotensin–aldosterone system. Plasma levels of ANP are increased in patients with heart failure caused by cardiac valve pathology, coronary heart disease, congenital heart defects, dilated and hypertrophic cardiomyopathies, and AF compared with control patients55. An analysis of the literature data suggests a relationship between AF and IAA58. Accumulations of amyloid fibrils in atrial myocardium of patients with AF surround and isolate cardiomyocytes and all interstitial components, interfering with contacts between cells, leading to their separation and disruption of electrical conductivity. They are a factor that provokes or supports AF.A change in the structure of the interstitium in atrial myocardium is one of the reasons for the development and maintenance of AF. The major morphological features of atrial myocardial remodeling in patients with AF are increased fibrosis, lipomatosis, and development of isolated atrial amyloidosis (IAA), which correlates with the age of patients with AF59 and myocardial sleeves of human pulmonary veins31. The authors have noticed heterogeneity in the distribution of TCs—they were not found in all patients and not in all samples17. In myocardium with significant sclerosis, a decrease in the number of TCs have been found60. TCs disappeared in the myocardium after a heart attack, but after a while, their number was increased in the peri-infarction zone, which confirmed the participation of TCs in tissue regeneration61.The morphological features of TCs at the ultrastructural level in pathologically altered myocardium is still unclear yet. In humans and mammals with AF, TCs have been detected in atrial myocardiumThe present study was focused on the analysis of the morphology of the left (LAA) and right (RAA) atrial appendages myocardium in patients with different forms of AF , persistent (PsAF) and long-standing persistent (LSAF) with special emphasis on the study of ultrastructural features of interstitial cells—TCs in two groups of patients: with AF and AF + IAA. We expected to define TCs function and identify these cells using immunohistochemical markers. The data obtained were compared with the morphological parameters of the myocardium and the clinical parameters of patients. We suggested that in patients with different forms of AF hemodynamic overload provokes various types of pathological remodeling of atrial appendage myocardium and expected to make a comparative analysis of the increase in fibrosis, lipomatosis, IAA and hypertrophy of cardiomyocytes. Moreover, we hypothesized that interstitial tissue reorganization can initiate the transformation of TCs with the appearance of “transitional forms” combining the morphological and immunohistochemical characteristics of TCs with signs of fibroblast-like cells, histiocyte-like cells, and endotheliocytes.p < 0.05). In patients with LSAF the length of cardiomyocytes significantly exceeded that in patients with PAF  and PsAF  (Table p = 0.0096). In the LAA myocardium of 25–50% of patients and in the RAA myocardium of 33–45% of patients with different forms of AF, cardiomyocytes with partial loss of myofibrils (myolysis) were detected. The presence of myofibril free zones negatively affected the contractile activity of cardiomyocytes, since a large proportion of the sarcoplasm of these cells did not contain myofibrils and was filled with glycogen granules and mitochondria. Nevertheless, the proportion of cardiomyocytes with myolysis did not differ between patients of various groups with AF  Table  and corrAF Table .Figure 1p = 0.015) and the RAA  myocardial remodeling .In patients with AF accumulations of Sirius red-stained amyloid deposits were detected in the interstitial area on the surface of cardiomyocytes sarcolemma and at the walls of the intramural vessels of atrial appendage myocardium Fig. c,d. An ip = 0.038) and positively correlated with the presence of IAA . Areas of sarcoplasm without myofibrils were mainly detected in LAA cardiomyocytes of small diameter  in patients with progressive dilatation of the left atrium , mitral valve regurgitation  and a shorter AF duration . Furthermore, in patients with PAF and IAA, cardiomyocytes with myolysis were less common , in turn, IAA was more pronounced in patients with a long duration of AF . In patients with PsAF, an increase in left atrial volume positively correlated with IAA , but inversely with LAA myocardial fibrosis  and IAA inversely correlated with fibrosis . LAA cardiomyocytes with myolysis were detected in PsAF patients with long AF duration . In LSAF patients, an increase in the diameters of LAA cardiomyocytes positively correlated with an increase in the left atrium size , dilatation of the fibrous ring of the mitral valve , and an increase in RAA cardiomyocytes diameters positively correlated with dilatation of the fibrous ring of the tricuspid valve . In contrast to the data for the PAF group, in patients with LSAF, an increase in the diameter of RAA cardiomyocytes was accompanied by the loss of myofibrils in them , moreover, cardiomyocytes with myolysis were more common in the myocardium with IAA .The above morphological characteristics of the myocardial interstitium and cardiomyocytes did not differ between AF groups, with the exception of the length of the cardiomyocytes Table . At the When dividing patients with AF into two groups according to the presence of IAA. the above indicated morphometric measurements of the proportion of fibrosis, the diameters and the lengths of cardiomyocytes, proportions of patients with myolysis in cardiomyocytes did not differ significantly in LAA and RAA myocardium of patients with AF and AF + IAA Table .Light microscopy, using semi-thin sections and TEM investigations of atrial appendage myocardial samples allowed us to identify TCs in the myocardium of patients of all AF groups by their morphology and location in the interstitial area Fig. a. It wasp < 0.05). In addition, in LAA myocardium it was significantly lower compared to RAA myocardium   and with a high degree of mitral valve regurgitation   Fig. f, and co33) Fig. g,h, whicThe TCs processes surrounded and “accompanied” the interstitial vessels of all types: they were located around the layer of capillary endotheliocytes, the layer of smooth muscle cells of small venules and arterioles, and were found in the perivascular zone next to large vessels Fig. a–c. AtriA comparative TEM analysis of the morphological features of TCs and fibroblasts demonstrated that despite the similarity of their localization in the interstitium, the ultrastructure of these cells differed greatly. The pathognomonic feature of TCs morphology was the presence of thin Tps with a diameter of 0.1–0.2 µm with local dilations. The diameter of TCs in the nucleus region was smaller than that of fibroblasts. Furthermore, significantly fewer rough reticulum (rER) tanks were found in the cytoplasm of TCs than in the cytoplasm of fibroblasts Fig. a,b. TEM p = 0.016) and high degree of tricuspid valve regurgitation . Besides, in both groups of patients with AF, TEM revealed centrioles in TCs  and negatively with IAA . In LAA, centrioles in TCs were detected in patients with a high degree of mitral valve regurgitation .The ultrastructural features of TCs did not differ in patients with various forms of AF, however, small differences were found between the myocardial TCs of LAA and RAA appendages. In LAA myocardium of 82.1% patients and in RAA myocardium of 68% patients with AF, TCs contained a large number of rER in the cytoplasm, including dilated ones, which indicated a high secretory activity of TCs Fig. d. In RAATCs Fig. e: in LAA32. At the same time, caveolae on opposite membranes of TCs and cardiomyocytes, exosomes, and multivesicular bodies located nearby in interstitium were probably related to the paracrine interactions between both types of cells , fibroblast-like cells (vimentin+), hematopoietic stem cells (CD34+) and cell adhesion protein (CD44+), that mediated the adhesion of hematopoietic, fibroblast and glial cells with the extracellular matrix. No differences were found between the TCs immunophenotypes of patients with various forms of AF as well as between the AF and the AF + IAA groups. CD117, vimentin, CD34, and CD44 positively labeled TCs were small-sized, spindle- or star-shaped. They were located in the interstitium next to cardiomyocytes, small and large vessels   Fig. i. The nu05) Fig. j. Thus, 9. The present study has analyzed the ultrastructural and immunohistochemical features of TCs in atrial appendage myocardium of patients with various forms of AF and found no differences between them. TCs size allowed us to accurately identify them only at the ultrastructural level by the main morphological feature—the presence of long thin Tps surrounding cardiomyocytes, blood vessels, and nerve fibers. The diameter of TCs, according to our data, did not exceed 4 μm and the diameter of TCs processes was 0.1–1 μm. Tps were up to hundreds of microns in length with dichotomous branching and local dilations, they formed a branched network. Such a morphology features coincide with the literature for the myocardial TCs of human and another mammalian in vivo and in vitro62.TCs are a unique type of connective tissue cells. Thin long processes of TCs coordinate all cells of the cardiac interstitium and provide a connection between them, although TCs occupy only about 1.2 ± 0.3% of myocardial volume and 0.5–0.7% of interstitial tissue44. Our morphometric study demonstrated in myocardium of patients with all forms of AF cardiomyocyte hypertrophy, with a partial loss of myofibrils (myolysis) in them followed by filling of free zones of sarcoplasm with glycogen granules and mitochondria, as described earlier39. In addition, patients with LSAF showed a significant increase in the length of the LAA cardiomyocytes compared to PAF and PsAF, which could explain the LA dilatation specific for AF.Atrial myocardium of patients with AF undergoes hemodynamic overload which leads to morphological changes in the interstitial tissue—an increase in the area of interstitial and perivascular fibrosis, disruption of tissue architectonics, and separation of interstitial cells59. The accumulation of amyloid fibrils is thought to disrupt the formation of adequate intercellular contacts, electrical conjugation of neighboring cardiomyocytes and paracrine interactions of TCs with interstitial cells surrounding them. IAA serves as a morphological substrate for the development, promotion, and maintenance of AF. According to other authors, IAA has been found in atrial myocardium of 9.6–39% patients with valvular defects and persistent AF58 and reaches 60.89% of patients43. The inverse relationship is also true—in patients with IAA, AF has been revealed in 48.5–82.35% of cases58. According to our data, patients with AF + IAA were older and had a greater degree of mitral valve regurgitation than patients with AF , MIP-2 and MCP-163, angiogenic microRNAs38. For example, through exosomal miRNA-21-5p, cardiac TCs ensure the survival of microvascular endothelial cells in myocardial infarction64.The long thin processes of the TCs (Tps) “accompany” all the components of the interstitium—cardiomyocytes, vessels of all types, nerve fibers. Interestingly, according to our observations, in atrial appendage myocardium of patients with AF, Tps were always located at some distance from other cells, suggesting paracrine regulation of their activity. The distance from Tps to the surface of cardiomyocytes and nerve fibers reached 0.9 μm, which slightly exceeded the data of other authors65. Transfer of TCs exosomes into infarct-affected rat myocardium attenuates cardiac fibrosis, improves cardiac function, and increases angiogenesis36. Similarly, in vitro cultivation of endotheliocytes in the presence of exosomes isolated from rat myocardial TCs stimulates proliferation, migration, and the formation of capillary-like structures by endotheliocytes36.TCs act as immunoregulators expressing toll-like receptors TLR 2, TLR 3, TLR 532. Some authors suggest that in children's myocardium, TCs are a component of the stem cell niche, provide their maturation, directed migration to the sites of differentiation. TCs take part in the formation of correct three-dimensional pattern of myocardial tissue37. In adult myocardium of patients with AF, TCs seem to perform other functions and their interaction with stem cells is impaired or not carried out.In atrial appendage myocardium of adult patients with AF, we did not found contacts of TCs with myocardial stem cells, previously described in children with congenital heart defects60. One of the most probable causes of lipid accumulation in the cytoplasm of TCs we believe activation of phagocytic activity of these cells, the possibility of which is confirmed in studies of CD34-positive TCs of the colon and skin66. We assume that remodeling of atrial myocardium with an increase in fibrosis and accumulation of amyloid fibrils in the interstitium progresses in adult patients with AF and leads to dystrophic changes in cardiomyocytes, their destruction, and, as a result, enhanced phagocytic activity in interstitial cells, including TCs.In this work, the ultrastructural changes of the atrial appendages myocardial TCs of adult patients with AF and AF + IAA were described in detail. We revealed, for the first time, lipid inclusions in the cytoplasm of some TCs, which formed conglomerates and sometimes filled almost the entire perinuclear zone. In other cases, single lipid drops were found in the perinuclear zone and dilations of Tps. Such changes in TCs ultrastructure corresponded to changes in their functions. Lipid inclusions interfered with the intracellular transport of substances in the cytoplasm, disrupted the synthetic function, and led to their inferiority. An analysis of the literature data regarding the ultrastructural features of TCs in the myocardium of patients with cardiovascular pathology has not found similar references. Some authors note the compression and shortening of Tps and the appearance of vacuoles in the cytoplasm (without identifying the cause of these changes), while the accumulation of lipid drops have not been described67, migrate from myocardial epicardia, where they acquire the properties of interstitial perivasular fibroblast-like cells. It is important to consider the differences between TCs and fibroblasts, the main cellular components of the interstitium. At the ultrastructural level, the difference in morphology and hence the function of these cells is obvious. Fibroblasts, unlike TCs, have thick short processes without dilations, a significantly large volume of cytoplasm, in which rER tanks predominate. Fibroblasts rarely contain caveolae and lack a basement membrane. They are often surrounded by collagen fibers. The main function of fibroblasts is the production of extracellular matrix components and control for the synthesis and degradation of its components. While TCs, having a branched network of Tps, apparently perform signaling functions and regulate the activity of all cellular components of the interstitium13. TCs differ from fibroblasts in the expression patterns of several genes69 and when cultured in vitro TCs are characterized by a higher ability for adherence, spreading, and Tps extension than fibroblasts70. In the present work, we were not able to register contacts between TCs and fibroblasts or to find a reliable relationship between TCs presence and the degree of fibrosis. Nevertheless, we noticed that the number of TCs was reduced in areas of fibrosis, which was probably due to their transformation into fibroblast-like cells. Similarly, Mitrofanova et al.59 indicates that the number of CD34+/S100+ TCs inversely correlates with the area of fibrosis and lipomatosis in the myocardium of patients with long-term persistent AF. In patients with systemic sclerosis, the content of CD34+/CD31- TCs in the left ventricular myocardium and lungs is significantly reduced compared with control samples27. In the myocardium of patients with end-stage heart failure caused by idiopathic dilated cardiomyopathy, ischemic and inflammatory cardiomyopathy and aortic valve stenosis the number of C-kit+ TCs/mm2 is also decreased more than twofold compared to the intact myocardium and inversely correlates with the amount of mature fibrillar collagen type I and collagen propeptides PINP and PIIINP60. In the zone of experimentally induced rat infarction, CD34+ and C-kit+ TCs are either not determined61 or their number is significantly reduced33.There is no reliable data on the origin of either telocytes in the literature. It can be assumed that TCs are pro-epicardial origin and of a mesenchymal nature, like other epicardial cells, undergo epithelial-mesenchymal transformation+), fibroblast-like cells (vimentin+), endotheliocytes (CD34+), fibroblast, and glial cells (CD44+). While marker of cells with neurogenic differentiation (S100+) was observed in nerve fibers, and the marker of activated endotheliocytes (endoglobin+/ CD105+) was found only in endotheliocytes, but not in TCs. Immunohistochemical markers listed above have been described in the literature and generally accepted for the detection of myocardial TCs71. Since none of these markers is specific, a number of authors suggests using double immunohistochemical staining for identification of TCs: CD34+/CD31-, in contrast to CD34+/CD31+ endothelial cells29, CD34+/PDGFR-α+10, CD34+/S100+59, as well as C-kit+/vimentin+36. The literature actively discusses the possibility of inaccurate identification of TCs at the light-optical level using different markers of IHC staining, in particular, during the differentiation of epicardial TCs, among which there may be cells expressing markers of lymphatic endothelial cells72. Thus, the absence of specific markers of TCs forces researchers to use various markers to identify these cells and, in our opinion, indicates the heterogeneity of their population or their ability to differentiate into other cell lines.In this work, in atrial appendages myocardium of patients with AF by immunohistochemistry we revealed interstitial cells, which, taking into account their location and morphological features could be attributed to TCs. These cells corresponded to immunohistochemical signs of stem cells  by TCs in adult atrial appendage myocardium. We have shown that CD68-positive histiocytes and TCs are more common in the myocardium of patients in the AF + IAA group than in the AF group. Probably, during myocardial remodeling in the AF + IAA not only the number of histiocytes but also the number of TCs-like “transitional cells” with histiocytic function increases in the interstitium. This is, in our opinion, a manifestation of compensatory mechanisms, which are expressed by a change in the structure and functions of these interstitial cells during arrhythmogenic myocardial remodeling complicated by age-dependent IAA. The mechanism of transition of one cell type to another type of cells through "transitional" forms has been demonstrated in the cell culture74. Apparently, this is not well understood fundamental biological phenomenon that provides compensatory and adaptive processes in tissue and organ. However, in order to confirm this, an integrated approach should be used, including the analysis of ultrastructural features of cells.The results obtained by us and discussed by other authors indicate that TCs are a highly plastic cell population. TCs properties and immunophenotype vary depending on the type of organ and the functional state of its interstitial tissue75. Other authors in the human myocardium identified more than 20 cell subtypes, which differed significantly by cardiac chamber, laterality, and gender76. In the above studies, there is no mention of TCs and it seems difficult to determine to which type of cells they can be attributed, since TCs simultaneously express markers of different types of cells—fibroblast-like, endotheliocytes, and, as we have shown, histiocyte-like. In addition, it can be assumed that the cell transcriptome may change depending on the microenvironment and composition of the extracellular matrix. We believe that none of the methods for studying isolated cells or their nuclei, including transcriptome analysis, can provide a complete picture. In this regard, morphological and especially ultrastructural identification of cells is very important. Popescu et al.77 suggested using the electron microscopy as a "platinum" standard for identification of TCs, including the analysis of the cell membrane, the presence of caveolae, mitochondria, endoplasmic reticulum, the number and thickness of processes, their location, and the presence of contacts with other cells.Currently in the human myocardium by the method of single-cell and single-nucleus RNA sequencing, in accordance with the analysis of myocardial cells transcriptome, the following 11 major cell types were identified: atrial and ventricular cardiomyocytes, fibroblasts, endothelial cells, pericytes, smooth muscle cells, immune cells (myeloid and lymphoid), adipocytes, mesothelial cells and neuronal cells+, CD16+ transitional forms of TCs we found in the atrial appendage myocardium of patients with AF is similar to the previously described morphology of cardiac resident macrophages 79. According to Hulsmans et al.79, these spindle-shaped, CD68+ and CD163+ resident macrophages with long processes that contact each other and with stromal cells are much more numerous in the human AV node than in the working myocardium, they express genes involved in electrical conductivity. In addition, M2 macrophages are involved in the regeneration of the heart when it is damaged, they are necessary for the proliferation of cardiomyocytes and are involved in angiogenesis80. The method of single nuclear RNA-sequencing of the transcriptome of human myocardial cells has shown that tissue-resident cardiac M2 macrophages associated with cardiovascular remodeling are characterized by expression of the lymphatic endothelial hyaluronan receptor LYVE1, scavenger receptors CD163 and COLEC12, the mannose receptor ubCH1, the E3 and liquin natural resistance-associated macrophage protein 1 (NRAMP1 or SLC11A1). Further isolation of subclusters revealed two more populations of cells that both express M2-polarization associated genes, including RBPJ and F13A1 and the transmembrane collagen COL23A176. Hulsmans et al.79 do not exclude the possibility of the participation of M2 macrophages in conduction abnormalities not only in the AV node, but beyond the AV node, including AF. However, what cells did the authors79 describe in his work—macrophages or TCs—is still a subject of debate. Given the complexity of the classification and accurate identification of these cells in the myocardium, we assume that the TCs described in our work are a “transitional form” of TCs with signs of resident macrophages. It is possible that the presence of a multiple immunohistochemical phenotype in TCs does not indicate a complete change in the direction of their differentiation, but reflects only the transition from one stable state of differentiation to another in the pathologically altered atrial myocardium in AF. A similar concept is discussed in the article81, where instead of the state of terminal differentiation of cells, it is proposed to consider the state of stable differentiation. We hypothesize that this adaptive mechanism may be activated in the interstitium of the remodeled atrial myocardium in aged AF + IAA patients with hemodynamic overload and AF.The phenotype of CD68We have proposed the following model describing the role of TCs in a pathologically altered myocardium in patients with AF. Hemodynamic overload of the left ventricle due to mitral valve defect leads to remodeling of the left atrial myocardium, the development of fibrosis, lipomatosis, an increase in the size of the atrium and includes a compensatory mechanism associated with the active production of ANP by cardiomyocytes. In patients with IAA, excessive production of ANP leads to its deposition in the interstitial area in the fibrillar form. Clusters of amyloid fibrils surround cardiomyocytes and all interstitial components, including TCs, isolate them and disrupt intercellular contacts. Under these conditions, there is an increase in phagocytic activity with an activation of synthetic function and proliferation of the TCs. Such compensatory changes in the structure and function of TCs are more pronounced in the myocardium of patients with impairment of valves function, long duration of AF. Changes in the morphology and, accordingly, TCs function are not the cause, but either the consequence of adaptation of the atrial appendage myocardium to function in AF + IAA states or part of the general age-related myocardial remodeling in AF + IAA. Disorganization of the regulatory function of TCs, along with IAA, probably provokes inadequate electrical impulse conduction and promote AF patients. Taken together, our results provide novel insight into cardiac TCs function, suggesting the universal mechanism for the development and maintenance of AF in the atrium with age-dependent IAA which is based on the disruption of contacts between cardiomyocytes and stromal cells, one of the most important components of which are TCs and the transformation of TCs with the acquisition of the properties of histiocyte-like cells.+, vimentin+, CD34+, CD44+, S100-, CD105- immunophenotype. No differences in morphology and immunophenotype of myocardial TCs were found in patients with various forms of AF. Immunohistochemistry also demonstrated the phagocytic activity of TCs-like cells in atrial appendage myocardium . CD68-positive interstitial cells were detected predominantly in the AF + IAA group than in the AF group. We suppose that in aged AF + IAA patients hemodynamic overload and pathological remodeling of atrial myocardium initiates the transformation of TCs, with the appearance of “transitional forms” combining morphological and immunohistochemical signs of fibroblast-like cells, histiocyte-like cells, and endotheliocytes. All of the above allows us to speculate about the undisclosed mechanisms of cellular plasticity, which accompanies the processes of age-related and pathological compensation and adaptation of organs and tissues.The present study confirms an increase of interstitial and perivascular fibrosis and lipomatosis and age-related development of IAA in atrial appendage myocardium of patients with PAF, PsAF, LSAF. IAA was an age-related factor of the AF myocardial remodeling and was detected in 4.3–25% biopsies of LAA and 21.7–41.7% biopsies of RAA myocardium. TCs were identified in atrial appendages myocardium of patients with all forms of AF, but more often in the AF + IAA group than in the AF group. In LAA myocardium, TCs presence correlated with the duration of AF, the degree of age-depended IAA, and also with a high degree of mitral valve regurgitation. However, there was no correlation between the TCs presence and the degree of fibrosis and cardiomyocyte hypertrophy. Ultrastructural signs of the phagocytic activity of myocardial TCs of adult patients in combination with the synthetic and proliferative activity were described for the first time. Adult atrial appendage myocardial TCs corresponded to CD117This study has limitations associated with estimating the number of TCs since the most accurate identification of these cells is possible only at the ultrastructural level. It is not possible to take a large biopsy sample without the risk of harming the patient during surgery. Tissue processing requirements for TEM also limit the size of the biopsy. Another significant limitation of studies on human myocardium by TEM is the lack of comparative studies in the intact heart associated with the requirements for immediate tissue fixation to avoid autolysis. Therefore, we had to use the atrial appendage myocardium of patients with AF as a comparison group for the patients with AF + IAA.Additional researches will be needed to expose the role of TCs in atrial myocardial remodeling in patients with AF. Moreover, further TEM investigations are required to determine the function of TCs and the directions of their differentiation in pathologically altered myocardium of patients suffering from cardiovascular diseases.p < 0.05). No patients with diabetes were found. 30–47.8% of patients with different forms of AF have suffered from arterial hypertension (2nd–3rd stage). Previously, only 4 patients (one from the PAF group and three from the PsAF group) underwent radiofrequency ablation of pulmonary vein.Human cardiac myocardial samples from left atrial appendage (LAA) and right atrial appendage (RAA) were collected from 55 adult patients (aged 24–71 years) with AF, documented by electrocardiographic monitoring , 20 patients with persistent AF (PsAF), 23 patients with long-standing persistent AF (LSAF). The duration of the arrhythmic history was from 6 months to 27 years. All patients underwent the modified radiofrequency maze procedure («Labyrinth 3B») between May 2016 and December 2017 at A.N. Bakulev National Medical Research Center of Cardiovascular Surgery, the Ministry of Health of Russian Federation, Moscow, Russia. Intraoperative biopsies were taken as part of the research process. All patients before operation were evaluated by transthoracic echocardiography and computed tomography of the left atrium. Table —53.1 ± 9.8 (24–66 years) . AF + IAA patients showed a high degree of mitral valve regurgitation and were more likely to suffer from arterial hypertension, than patients in the AF group . The AF + IAA group included patients with all forms of AF, presented in almost equal proportions , while the AF group predominantly consisted of patients with LSAF (52%) and PsAF (33%) forms of AF.According to the results of histological examination of the atrial appendage myocardial biopsy at light microscopy  and TEM (determination of deposits of amyloid fibrils), patients were divided into 2 groups: patients with AF and signs of IAA  and patients with AF without signs of IAA  . Investigation of the patients' biological materials was legally confirmed by the patients' informed consent.—no deposits; 1—deposits were around single cardiomyocytes; 2—around small groups of cardiomyocytes; 3—around half of cardiomyocytes and single vessels; 4—around more than 75% of cardiomyocytes and blood vessels. The slides were examined by light microscope Leica DMRB with Leica DFC495 camera and Leica PL FLUOTAR 10 × /0.3 and Leica N PLAN 40 × /0.65 .Myocardial fragments were fixed in 4% buffered formalin, processed by a standard technique, and paraffin-embedded sections (3–4 μm thick) were stained with hematoxylin and eosin, Masson’s trichrome (Bio-Optica), Sirius Red (Bio-Optica) and used for histological examination. Masson’s Trichrome-stained sections were used in total for morphometric evaluation of percent fibrosis at × 100 using the Image-Pro Plus program . Sirius red-stained sections were used for a semi-quantitative 5-point assessment of the amyloid deposits distribution in the interstitium around blood vessels and cardiomyocytes: 0—the zones without myofibrils occupied 10% (0), 10–50% (1), 50% (2) and more than 50% (3) of sarcoplasm.Atrial appendage biopsies collected from all patients were also processed for thin-section light microscopy and TEM. In brief, small fragments of the myocardium were pre-fixed in 2.5% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), post-fixed in 1.5% osmium tetroxide solution, dehydrated in graded concentrations of alcohol and embedded in Araldite. Semithin sections (1 μm thick) were stained by the Periodic acid–Schiff (PAS) method with additional methylene blue staining. The diameters of cardiomyocyte were measured passing through the nucleus in at least 50 cells for each patient . In the same cardiomyocytes, the degree of myofibrils loss (myolysis) was determined on a 4-point scaleUltrathin sections were cut using ultramicrotome Leica ultracut UCT . Ultra-thin sections (50–70 nm) were collected on copper grids, contrasted with uranyl acetate and lead citrate, and examined using a Philips CM100 transmission electron microscope . To assess the presence of the TCs at the ultrastructural level (× 4800), a semi-quantitative 6-point scale was used: TCs and their processes were absent (0); there was at least one TC in one field of view (1); several TCs and their processes were in less than half of the fields of view (2); in half the fields of view (3); in most fields of view (4); in all fields of view (5). TCs and prolongation diameters were measured at ultramicrophotographs (× 4800).For the immunohistochemical study, formalin-fixed paraffin-embedded tissue sections (3–4 μm thick) were mounted on adhesion slides . Deparaffinized and dehydrated sections were rinsed in phosphate buffer solution (PBS) at pH 7.4, incubated in hydrogen peroxidase block solution at 37 °C for 15 min, washed twice in PBS. Then 0.01 M citrate retrieval solution (pH 6.0) at 90 °C for 20 min were used. After that sections were cooled to room temperature for 20 min, washed twice in PBS and incubated with primary antibodies,as follows: CD117/C-kit rabbit, monoclonal,ready-to-use , vimentin rabbit, monoclonal, ready-to-use , CD34 mouse, monoclonal, ready to use , CD44 mouse, monoclonal, 1:50 , CD68 rabbit, monoclonal,ready-to-use , CD16 rabbit, monoclonal, 1:100 . Then sections were treated with secondary anti-mouse and anti-rabbit antibodies with streptavidin–biotin complexes  following standard manufacturer's instructions. After that sections were washed twice in PBS and counter-stained with hematoxylin. The positive reaction products were detected by brown staining of the cells. Negative control was obtained by omitting the primary antibodies in the standard immunohistochemical protocol. The slides were examined by light microscope Leica DMRB with Leica PL APO 63 × /1.4 oil. Representative photomicrographs were taken using and Leica DFC495 camera . The numbers of CD68 and CD16 immunopositive cells in the interstitium were counted (× 400) over the whole histological sections and presented as a fraction of the total number of cells.For ANP/desmin double immunofluorescence reaction paraffin-embedded tissue sections were deparaffinized and dehydrated. Then sections were treated with 0.01 M citrate retrieval solution (pH 6.0) at 90 °C for 20 min, cooled to room temperature, washed twice in PBS and incubated with 5% bovine serum albumin (SAFC A3059) and 0.5% Triton X-100  in PBS for 60 min to block non-specific binding sites. Then sections rinsed in PBS and incubated with a cocktail (1:1) of primary antibodies for 90 min at 37 °C: antibody to the Atrial Natriuretic Peptide, mouse monoclonal,1:300  and desmin, rabbit monoclonal 1:500 . After washing in PBS sections were incubated with a cocktail (1:1) of two secondary antibodies conjugated with fluorochromes: Alexa 488, goat anti-rabbit 1:300  and Alexa 546, goat anti-mouse 1:300 . The nuclei were stained with 4′, 6-diamidino-2-phenylindole . Immunofluorescence-stained preparations were embedded in Immu-mount (Thermo-Shandon) and examined using confocal laser microscope Leica TCS SPE .p < 0.05. The associations between TCs presence, their ultrastructural and immunohistochemical features and clinical and morphological parameters of the myocardium of patients with AF were determined using the non-parametric Spearman correlation coefficient at p < 0.05 .Statistical analysis was performed using the Statistica 10.0 software program package. The corresponding clinical data of patients and myocardial morphometry data obtained through research myocardium of patients with different forms of AF as well as between AF and AF + IAA groups were compared using the non-parametric Mann–Whitney test. Differences between groups were considered significant at


Animals born with physical impairments may particularly require behavioural flexibility and innovation to survive and carry out social activities, such as grooming. Studies on free-ranging Japanese macaques on Awaji Island, Japan, have shown that individuals with congenital limb malformations exhibited compensatory behaviours while grooming, such as increased mouth and elbow use for removing ectoparasites. The aim of this study is to explore disabled and nondisabled grooming techniques to determine whether and to what extent disabled monkeys develop novel grooming techniques, and if there is disability-associated variation in grooming efficiency. We hypothesized that modified grooming techniques used by disabled monkeys fulfilled the social and relaxing functions of grooming, however, that grooming by manually impaired individuals may still carry a hygienic cost to the recipients. Grooming behavioural data were collected by video in 2007 on 27 adult females (11 with CLMs). With a detailed grooming-related ethogram, we transcribed 216 2-minute continuous grooming video samples. We analyzed the data using generalized linear mixed effects models in R. We found that monkeys with manual impairment were less efficient groomers, as measured by removal and movement efficiency during grooming. However, there were no significant differences associated with the number of grooming movements per sample among the focal animals. Additionally, with a behavioural sequential analysis, we isolated 8 distinct grooming techniques and 3 novel disability-specific movements. Our results indicate that innovation and modification of movement types does not entirely compensate for manual disability, and that manual impairment carries a cost to the hygienic function of grooming. However, for the grooming recipient, the experience of being groomed by a disabled or nondisabled groomer is likely similar, and through movement compensation, disabled monkeys are able to engage in the social aspect of grooming without incurring any disability-associated costs. Free-ranging and wild animals are exposed to many environmental and social challenges, changes and conditions that can lead them to modify their behaviours or innovate novel behaviours in order to survive and reproduce . Such beHowever, animals are often able to compensate behaviourally for physical impairment. Examples of compensatory behavioural responses were observed in fruit flies that, after leg-amputation, recovered their unbiased walking . Ang andA group of Japanese macaques with a high instance of individuals born with limb malformations, presents an opportunity to examine the influence of congenital impairments on the behaviour of free-ranging animals. The cause of these malformations of the limbs and digits remains unclear; however, the population is small and isolated, and the presence of pesticide residues in provisioned food has been suggested as a contributing factor involved in the occurrence of such malformations ,13. In sSocial grooming or allogrooming (hereafter grooming) in non-human primates and other animals has a hygienic function, lowering ectoparasite burdens, as well as a social function associated with the maintenance of social group cohesion –18. AddiIn nondisabled Japanese macaques, louse egg handling techniques that occur during grooming  have been found to vary according to maternal kinship . These kIn this study, we address the following research questions: 1) Are primates with extensive manual impairments able to compensate for their disabilities during allogrooming, and if so, how do they compensate? 2) Are disabled female Japanese macaques able to fulfill the hygienic function of grooming, assessed as grooming efficiency ? 3) Are disabled female Japanese macaques able to fulfill the social and relaxing functions of grooming, as measured through frequency of grooming gestures used? 4) Do disabled females employ novel grooming techniques and/or innovate novel grooming gestures? 5) Is there a difference in the number and variety of grooming techniques used by disabled and nondisabled females?We hypothesized that disabled females would show significant differences in their grooming behaviours in comparison to nondisabled females, and we expected that disability would carry a cost in terms of manual dexterity and would affect the grooming techniques used by disabled individuals, as well as their grooming efficiency. However, we also hypothesized that manually impaired females would be able to compensate behaviourally for their physical impairments. In particular, while we expected that the movements developed by disabled individuals would not meet the hygienic efficiency of nondisabled females, we expected that they would use a similar gesture rate in grooming, which would allow them to fulfill the social function of grooming. Evidence of behavioural flexibility could emerge from our results in a number of ways: a finding of no significant difference in grooming efficiency, with substantial variation in grooming techniques could suggest that behavioural flexibility compensates for disability caused by CLMs; individuals could also demonstrate behavioural flexibility through the innovation and use of novel grooming techniques, or through modification of commonly used grooming gestures and sequences. In any case, we would expect to see a wider variation in grooming techniques among disabled compared to nondisabled individuals.All data collection was non-invasive and in accordance with the requirements of the University of Calgary Animal Care Committee (Protocol number: BIO8R-03) and those of the Awajishima Monkey Center in Japan, with video data collection protocols that have also been approved by the Animal Use Committee at Concordia University (Protocol number 30009663).The Awajishima Monkey Center (AMC), located on Awaji Island in the Seto Inland Sea of Japan, is a privately-owned tourist destination and field research station with a free-ranging and provision-fed population of ~400 Japanese Macaques . PhysicaThe videos were recorded at the AMC from May to July 2007, using a Sony camcorder. Videos were collected with the goal of obtaining continuous focal animal data . Each foTo analyze the videos, we created a grooming behavioral ethogram including movements performed by disabled and nondisabled Japanese macaques , it was not always possible to see the egg itself in the video . However, since removing a louse egg involves the use of fine motor skills (gripping the egg and doing a pinch) or precision use of the mouth, individuals often required several attempts to successfully complete this action. For these reasons, we defined the following criteria for identifying a successful louse egg removal when the egg itself was not visible on the video: when the subject used her hand to detach the egg and bring it to her mouth, or used her teeth directly to detach the egg, the gesture had to be followed by a change in the groomed area to be counted; if the subject licked the area instead of using her teeth, this was not counted as an egg removal.A total of 216 2-minute video focal grooming samples were analyzed. For assessing the grooming efficiency of the subjects, we recorded the number of grooming movements and the number of eggs removed per sample. We calculated the average number of movements performed per egg removed and determined how often the mouth was used to remove an egg. Infant-related behaviours and other behaviours were not included in the analysis  to analyze the quantitative grooming data . Mixed eWe ran three sets of models to test our hypotheses. We ran each set of models with manual physical impairment as the fixed factor and individual focal monkey as a random factor. Each model was run using a categorical measure of disability (disabled or nondisabled) and a continuous measure of manual disability  as fixed factors  or the probability of doing Y after X (lag 1) was higher than 15% . On the We evaluated the flowcharts qualitatively to identify grooming styles and the use of disability-specific techniques. Finally, we used Mann-Whitney U-tests to determine if grooming efficiency varied significantly among grooming styles. For all statistical tests in this study, the results were considered to be statistically significant when P values were < 0.05.Full statistical results are available in Removal efficiency of nondisabled females was significantly higher than their disabled counterparts: on average, nondisabled females removed 71.45% more eggs than disabled females . Likewise, the extensiveness of manual impairment  also had a significant negative effect on removal efficiency; as the index value increased, the number of eggs removed decreased  .There was no significant disability-associated difference in the number of grooming movements performed per 2-minute sample .Movement efficiency was significantly and negatively associated with disability  and the index of manual disability . The results reveal that disabled females used more movements per egg removed than nondisabled conspecifics and that as the index of disability increased, the number of movements per egg removed increased as well .Disabled females used their mouth to groom significantly more frequently than their nondisabled counterparts  .We found that female Japanese macaques groomed in a predictable sequence, consisting of four stages performed in the following order: 1) Find the egg, which comprised all movements used for parting the hair and locating the egg; 2) Grip the egg, with the movements to loose, grip, and see the louse egg after its removal; 3) Carry egg to mouth, including the movements to bring the louse egg to the mouth; and 4) Eat removed egg, with a mastication movement . Based oMouth directly (md) gesture were 17.58% less movement efficient than those who did not . However, the removal efficiency was not significantly different between both groups   were observed to use the mouth directly for removing louse eggs in 13 out of 15,119 transition frequencies. As a result, we identified two distinct nondisabled grooming techniques associated with movement variation in the third stage: 1) the main nondisabled grooming technique (ND) and 2) the modified nondisabled grooming technique (NDM). Individuals that used the = 0.155) .Find the egg stage by using a Hand/arm push (hp) movement for parting the hair with one hand followed by a Finger sweep (fs) movement through the hair with the index finger of the opposite hand, to look for louse eggs. Then, she uses the Two digit pull (sp) movement to make the skin area more visible. While in this stage, she may use the Grab groomee’s limb (gl) movement to accommodate the groomee and inspect a different area. She will continue in this stage until she finds an egg to remove. Once an egg is located, the Grip the egg stage starts, she can use her nail for loosening the adhesive of the louse egg with a Thumb/nail loosening (nl) gesture or directly use her index and thumb to do a Two-digit pinch (dp) to remove the egg. Then, to confirm that the egg was removed from the hair, she checks her hand with a See egg on finger/hand (se) movement. If she observes that the egg is still attached to a pulled hair, she uses Second-hand support (sh) for detaching the egg and checks again if the egg is completely hair-free. Next, in the Carry egg to mouth stage, she brings the egg to her mouth with the hand holding the egg using the Single hand to mouth (sm) movement. Sometimes, she may look at her hand again to make sure the egg is no longer in her hand. Finally, at the Eat removed egg stage, she does a Louse egg mastication (em) gesture for eating the egg. After this, she goes back to the first stage either with a Finger sweep (fs) or a Two-digit pull (sp) if she continues grooming in the same area, or with a Hand/Arm push (hp) to continue somewhere else.In first nondisabled technique (ND) , the indMouth directly (md) movement in the Carry egg to mouth stage. After locating an egg, they may skip the Grip the egg stage and remove it directly with their mouths. The transitions going towards the Mouth directly (md) movement are dotted because its frequency was lower than 15%. The use of Nail loosening (nl) (10/16), Grab groomee’s limb (gl) (15/16), and the Second-hand support (sh) (14/16) movements were not observed in all nondisabled individuals, however the associated transition variations did not meet the criteria for defining a separate grooming technique.The second nondisabled technique is a modification of the main technique (NDM)  movement was common among all disabled groomers, and we classified it as disabled-associated gesture; although this gesture is used occasionally by nondisabled individuals, disabled monkeys used it significantly more than the nondisabled individuals . Additionally, the uals See .Hand/Arm push (hp) followed by Grab groomee’s limb (gl). This technique showed significantly higher movement efficiency than the ND technique , but was not significantly different from the NDM technique . There was no significant difference in the removal efficiency when compared with the ND technique , nor when compared with the NDM technique  (Table C in The first disabled technique DA  was usedTwo hand/arm to mouth (tm) movement in the Carry egg to mouth stage. In this technique, after gripping the egg, the groomer either used their mouth to remove the egg, brought a single hand to the mouth or, in the most frequent transition, they brought both hands or arms directly to the mouth, followed by the See egg on hand/ end of hand (se) movement, and finishing with a Louse egg mastication (em) movement. With this disabled technique DB movement efficiency was significantly lower than for nondisabled technique ND , but it was as efficient as the NDM technique . No difference in the removal efficiency was found when compared with the ND technique , nor when compared with the NDM technique .  movement and instead used the distil end of both arms for gripping the egg with a new gesture: Two hand/arm pinch (tp). Then, after looking at the egg on their hands, they frequently used Two hand/arm to mouth (tm), another new movement, at the Carry egg to mouth stage. In the same stage and with a similar frequency, they used the Mouth directly (md) gesture to remove and eat the egg. Also, although less frequently, they sometimes used a single hand to carry the egg to the mouth. The individuals using this technique had a significantly lower movement and removal efficiency compared to individuals using the ND technique  and the NDM technique   and 0.65 (Punch 98)) use the third disabled technique DC  movement to remove the louse egg and eat it. The individual using this technique had a significantly lower movement and removal efficiency compared to individuals using the ND technique  and the NDM technique   followed by the modified Hand/arm sweep (hs) in the Find the egg stage, and a Mouth directly (md) gesture to remove the egg. However, this sequence did not end with a Louse egg mastication (em) gesture because the female never followed the rule of changing the grooming area after using her mouth. In contrast, when using the new Two hand/arm pinch (tp) movement, followed by the novel Two hand/arm to mouth (tm), the individual checked if the removed egg was on her hand and then did the Louse egg mastication (em) movement, while changing the grooming area that confirmed the egg removal. In this technique, only 5 transitions were shared in common with the main nondisabled technique (ND). This technique had a significantly lower movement and removal efficiency compared to the ND technique  and the NDM technique   movement for parting the hair to find louse eggs. With the opposite arm, she performed a modified Hand/arm sweep (hs) to look for eggs in the groomee’s hair. Then, she always used the Mouth directly (md) gesture to remove the egg, followed by a Louse egg mastication (em) movement to eat the egg. This is the least complex of the grooming techniques since it comprises only four movements joined by eight transition lines of which none is common with the main nondisabled grooming technique ND. This technique has a significantly lower movement efficiency than the ND  technique. However, no significant difference was found with the NDM technique . Moreover, this disabled technique has a lower removal efficiency than ND  and NDM  . Moreover, efficiency decreased when the degree of manual disability increased, revealing that manual physical impairment impacts the ability to remove louse eggs, and that these costs were higher with more extensive physical impairments. These results correspond well with results from studies on manual disability in chimpanzees, where the most severely snare-injured individuals showed reduced feeding efficiency Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: I enjoyed reading this paper and appreciated the authors clarity with respect to overall organization, writing, and analysis. The authors provide an interesting contribution to the literature that focuses on behavioral flexibility and primates’ adaptation to disability. I suggest it be accepted for publication in Plos One. I proposed below a few recommendations for the authors to consider in any revision that may occur.Minor editorial suggestions:• Line 41 should the world “in” be “any”?• Lines 52 and 70 comma needed after e.g.? (you used commas after i.e.)• Line 80 macaque (lower case)• Line 152 order; however,• Line 163 samples (lower case)• Some inconsistencies in case for your ethogram behaviors. It appears that you are following the pattern of capitalizing the first word and lower case for the rest. With that in mind, please see lines 288, 316-319, 331, 341, 375, 388, 398, 399, 402, 415, 455, 487, 488, 498• Line 513 efficiency; however,• Line 546 I suggest you delete the word “properly”• Line 562 conditions • Lines 561-566 The information might read more clearly broken into two sentences, for example, at line 564 grooming. However,Analytical suggestions:N.B.: In my comments below, D=Disabled individuals, ND=Nondisabled individuals• Lines 542-544 some additional discussion of this might be helpful. It occurs to me that one response that a potential groomee might have to being approached for grooming by a D (and therefore less efficient) individual would be to refuse the invitation to groom. The source cited on lines 544-5 (14) may address this, but it was unclear to me whether you meant D individuals being invited to groom others, or D individuals being groomed. I am wondering if D individuals groom more frequently with relatives . I wondered whether D females had fewer grooming partners compared to ND females…I apologize if you reported this, and I missed it! But I couldn’t easily find it on my first or second read. I suspect that is the case and thought it might relate to your discussion of reduced efficiency by ND females.• An interesting characteristic of this population is that you have information on maternal relatedness. As I read your paper, I wondered how matrilineal relationships might impact on your data and/or on your interpretations of it. Lines 82 -87 raise the issue of technique being transmitted through kin lines. Are you arguing in the following sentences that trial and error learning overrides this? I felt like this could be discussed more thoroughly at lines 464-467 and specifically wondered whether your six ND females who used the mouth directly to groom were related to any of the D females. It would be helpful to indicate kin structure in S1 table, as I (again) wondered about the relationships among the females you studied. Lines 440-454 is another point in the manuscript where I had questions about kinship and how matriline membership might be (or might not be!) patterning the data. If possible, I think your arguments would be strengthened by including matriline as a variable in your analysis. It might also be helpful to indicate whether disabilities in this population are clustered within matrilines, or are disabilities distributed across the population?• Another compelling feature of your population is that you likely have more individuals living to be old than is true of unprovisioned populations. I did not see age reported in S1 table, and I suggest that be included. The disability classifications you used in this paper focused on those that influence manual dexterity/grip, but it occurred to me that visual impairment might be occurring in your older females . As was true for matriline membership, I wondered whether age explained any of the patterning in your dataset. If your ND mouth directly groomers are old females, that might account for their use of that technique. It seems that relying more on one’s mouth might occur as the ability to see the louse is reduced or lost.Thank you for this opportunity to review your interesting work.Reviewer #2: This manuscript presents a novel analysis of grooming technique in disabled Japanese macaques, taking advantage of a population with a high degree of disability to accomplish this. Theoretical background is multi-faceted and well researched. Methods are clear and well-suited to address their hypotheses. The results are presented clearly. Discussion covers needed areas well.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentPlos One Review.docxSubmitted filename: Click here for additional data file. 21 Jan 2020Reviewer #1:I enjoyed reading this paper and appreciated the authors clarity with respect to overall organization, writing, and analysis. The authors provide an interesting contribution to the literature that focuses on behavioral flexibility and primates’ adaptation to disability. I suggest it be accepted for publication in Plos One. I proposed below a few recommendations for the authors to consider in any revision that may occur.Thank you for the positive assessment of our research and for taking the time to provide us with your valuable suggestions, thoughts and feedback.Minor editorial suggestions:• Line 41 should the world “in” be “any”?Done (Line 41)• Lines 52 and 70 comma needed after e.g.? (you used commas after i.e.)Done (Line 52 and 70)• Line 80 macaque (lower case)Done (Line 80)• Line 152 order; however,Done (Line 153)• Line 163 samples (lower case)Done (Line 164)• Some inconsistencies in case for your ethogram behaviors. It appears that you are following the pattern of capitalizing the first word and lower case for the rest. With that in mind, please see lines 288, 316-319, 331, 341, 375, 388, 398, 399, 402, 415, 455, 487, 488, 498Done. Changes are located at lines 267, 268, 291, 319-322, 334, 378, 391, 401, 402, 405, 418, 458, 504, 505, 515. We applied this suggestion to all the behaviors listed in the ethogram in both the manuscript and the supplementary material S1.• Line 513 efficiency; however,Done (Line 530)• Line 546 I suggest you delete the word “properly”Done (Line 574)• Line 562 conditions Done (Line 591)• Lines 561-566 The information might read more clearly broken into two sentences, for example, at line 564 grooming. However, Done (Line 593)Analytical suggestions:N.B.: In my comments below, D=Disabled individuals, ND=Nondisabled individualsLines 542-544 some additional discussion of this might be helpful. It occurs to me that one response that a potential groomee might have to being approached for grooming by a D (and therefore less efficient) individual would be to refuse the invitation to groom. The source cited on lines 544-5 (14) may address this, but it was unclear to me whether you meant D individuals being invited to groom others, or D individuals being groomed.This is an interesting point. Unfortunately, we don’t have the contextual behavioural data to adequately address it here. That is, in the field at AMC, it is generally possible to identify when an animal is “presenting themselves for grooming” . We have data on the disability status of those who are involved in this “groom present” behaviour during focal follows and we detected no disability-associated differences (see Lines 569 – 571) for rewritten clarification of this point). What is more difficult to distinguish is if a monkey approaches another monkey with the intention to groom them . We are not able to reliably distinguish between approach-with-the-intention-to-groom versus a generalized approach . Also, approach-depart data were not collected in this dataset, so although we might otherwise examine the dynamics of approach-depart in relation to disability, that will have to await future studies . All this to say, yes, this would be so interesting to know, but at the moment, we don’t have the data to examine this question. However, based on your comments, we have now clarified the information we do have regarding groom-present behaviours and disability, as well as kin relationships in the text (Lines 558-573).• I am wondering if D individuals groom more frequently with relatives .In an earlier study, we found that for all females, available kin was positively associated with the number of grooming partners, however, kin availability had a stronger influence on number of grooming partners for disabled females, compared to nondisabled females. We’ve added a note about this to the new parts of the discussion on kin and grooming (Lines 561 – 562)• I wondered whether D females had fewer grooming partners compared to ND females…I apologize if you reported this, and I missed it! But I couldn’t easily find it on my first or second read. I suspect that is the case and thought it might relate to your discussion of reduced efficiency by ND females. Thanks. We did report this  but can see that it needed clarification. We have re-written this information to clarify and highlight it better (please see lines 558 - 569). We have integrated this information with other aspects of your suggestions, including new text in two parts of the discussion . Data for these social measures come from references 6 and 14. Disabled females in this group had fewer grooming partners but spent a similar amount of time being groomed compared with nondisabled individuals.• An interesting characteristic of this population is that you have information on maternal relatedness. As I read your paper, I wondered how matrilineal relationships might impact on your data and/or on your interpretations of it. Lines 82 -87 raise the issue of technique being transmitted through kin lines. Thanks for your comments. Yes, such a potentially interesting topic! We’re working on this now in another study. We have also added some text to the discussion to better address this possibility (please see above).• Are you arguing in the following sentences that trial and error learning overrides this? Not necessarily, we think it doesn’t override the transmission of techniques by kinship but complements it. In the case of disabled animals, we think that they imitate their relatives’ technique(s) and then develop individually appropriate styles through trial and error. Unfortunately our data on kinship were not detailed enough to allow for an in depth analysis of this topic, but based on your comments, we ran some tests . We are currently investigating the influence of kin and social network on grooming styles in a new study. Although we have some kin relationships in this study, we don’t have complete information: we had only 6 mother-daughter pairs and saw no obvious relationships, but the sample size was too small to draw significant conclusions.• I felt like this could be discussed more thoroughly at lines 464-467 and specifically wondered whether your six ND females who used the mouth directly to groom were related to any of the D females. Thanks, we added to the discussion in lines 472 – 482 to address this idea. 2 of the 6 nondisabled females have a disabled mother, 1 has a nondisabled mother, and there is no kin information about the other 3, so the sample size was too small to draw many conclusions. • It would be helpful to indicate kin structure in S1 table, as I (again) wondered about the relationships among the females you studied. Good idea. Thanks! We added a column in Table A in S1 with the maternal kinship information we had available, and a separate column for matriline.• Lines 440-454 is another point in the manuscript where I had questions about kinship and how matriline membership might be (or might not be!) patterning the data. If possible, I think your arguments would be strengthened by including matriline as a variable in your analysis. It might also be helpful to indicate whether disabilities in this population are clustered within matrilines, or are disabilities distributed across the population?When we were doing our initial analyses for this paper, we had put aside the idea of examining kinship because our kinship dataset is incomplete in various ways, and there are some females for whom kinship data are lacking. However, you raised some very good points, and so we explored this question further. We conducted a series of Kruskal-Wallis tests on some different metrics of kin availability . Then, separately, we conducted Kruskal-Wallis tests with matriline as a predictor variable. We also ran some linear models to see if there were any significant interactions occurring.We found no significant effect of matriline as a predictor of mouth use . However, we noted that all six of the nondisabled monkeys who used their mouths during grooming had disabled monkeys in their matrilines, only one nondisabled female who was known to have a disabled female in her matriline did not use her mouth while grooming. We have now added text to the discussion to highlight this information and clarify matriline and age as potential variables in the study The possibility that disability-associated grooming styles are transmitted within matrilines is interesting and we are currently investigating this question in the field, along with questions about social grooming network.• Another compelling feature of your population is that you likely have more individuals living to be old than is true of unprovisioned populations. I did not see age reported in S1 table, and I suggest that be included. Yes, good point. We have now added age to Table A in S1.• The disability classifications you used in this paper focused on those that influence manual dexterity/grip, but it occurred to me that visual impairment might be occurring in your older females . As was true for matriline membership, I wondered whether age explained any of the patterning in your dataset. If your ND mouth directly groomers are old females, that might account for their use of that technique. It seems that relying more on one’s mouth might occur as the ability to see the louse is reduced or lost.Thanks for this reference and suggestion. To find if Age had a significant effect on grooming efficiency, we conducted again the statistical tests including Age as fixed factor and all of them resulted in nonsignificant outcomes. In addition to that, we noted the use of Mouth directly (md) was observed in ND females of different ages (not only in old ones). The visual impairment hypothesis is an interesting idea, but our limited data do not support it at this point. However, this idea was added to the discussion (lines 467 - 471) for future work, and in the section on mouth use in grooming (see above).Thank you for this opportunity to review your interesting work.Dr. Sheeran, thank you so much for the comments, your thoughtful suggestions and encouragement, we hope that you will find the manuscript substantially improved. We appreciate your thoughts and feedback!Reviewer #2:This manuscript presents a novel analysis of grooming technique in disabled Japanese macaques, taking advantage of a population with a high degree of disability to accomplish this. Theoretical background is multi-faceted and well researched. Methods are clear and well-suited to address their hypotheses. The results are presented clearly. Discussion covers needed areas well.Thank you very much for taking the time to read our manuscript and for the positive assessment of our research.AttachmentResponse to Reviewers.pdfSubmitted filename: Click here for additional data file. 29 Jan 2020Social grooming efficiency and techniques are influenced by manual impairment in free-ranging Japanese macaques (Macaca fuscata)PONE-D-19-28479R1Dear Dr. Espitia Contreras,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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