Cancer remains as one of the leading cause of death worldwide. The development of cancer involves an intricate process, wherein many identified and unidentified factors play a role. Although most studies have focused on the genetic abnormalities which initiate and promote cancer, there is overwhelming evidence that tumors interact within their environment by direct cell-to-cell contact and with signaling molecules, suggesting that cancer cells can influence their microenvironment and bidirectionally communicate with other systems. However, only in recent years the role of the nervous system has been recognized as a major contributor to cancer development and metastasis. The nervous system governs functional activities of many organs, and, as tumors are not independent organs within an organism, this system is integrally involved in tumor growth and progression. Cancer is the leading cause of death worldwide due to the aging population and unhealthy lifestyle . AlthougStudies have demonstrated that the nervous system facilitates development of tumor metastasis by modulating metastatic cascades through the release of neural-related factors from nerve endings such as neurotrophins, neurotransmitters and neuropeptides –29. The Tumor metastasis initiation and clonal expansion is a complex process where contributing factors are not well understood. It is believed that metastasis process is initiated when genetically unstable tumor cells adjust to a secondary site microenvironment . This prAnoikis is a programmed cell death induced upon cell detachment from extracellular matrix, acting as a critical mechanism in preventing adherent-independent cell growth and attachment to unsuitable matrix, thus avoiding colonizing of distant organs , 37. ForEMT is a fundamental process for tumor progression by increasing invasiveness and resistance to anoikis and significantly elevating the production of extracellular matrix constituents leading to metastasis –43. EMT Nervous system modulates the function of vascular system which is essential for tumor cell extravasation and colonization. It has been found that neuropeptides such as substance P (SP) and bradykinin enhance vascular permeability promoting tumor cell extravasation and colonization , 29. In Development of tumor angiogenesis is essential for tumor growth and progression. Vascular endothelial growth factor (VEGF) plays significant role in tumor angiogenesis, leading to metastasis –47. StudThe nervous system plays a fundamental role in regulating immune responses . InflammTumor microenvironment  plays essential role in the formation of cancer metastasis. Stromal cells produce neural-related factors and express β-adrenergic receptor that facilitated tumor cell proliferation and survival in the primary site and secondary organ , 24. TumPerineural invasion (PNI) also known as neurotropic carcinomatous spread is a process mainly categorized by neoplastic invasion of the nerves. PNI is defined as the presence of cancer cells in the perineurium; it is believed to be a common route for cancer metastasis can cause cancer-related pain –68. The NTR receptors is associated with lymph node metastasis in a mouse model of breast cancer [PNI is influenced by the interaction between the nerve microenvironment and neurotrophic molecules expressed by cancer cells such as nerve growth factor (NGF), BDNF, glial cell line-derived neurotrophic factor (GDNF) and their receptors , 68, 71.t cancer . In OTSCt cancer , the pret cancer . Overexpt cancer . Converst cancer . GDNF hat cancer . Cancer-t cancer .BDNF facilitates cancer metastasis via binding to its receptors, TrkB/ TrkC and/or p75NTR as demonstrated in breast , colorecCollectively, these studies demonstrate that neurotrophins and their receptors play crucial role in PNI. These studies also suggest that the presence of PNI could be an effective predictor of metastatic potential and patient survival.Cancer-related neurogenesis (tumor innervation) is attributed to the ability of cancer cells to attract normal nerve fibers via the secretion of signalling molecules and neurotrophic factor. However, recent study has demonstrated that cancer stem cells are capable of directly initiating tumor neurogenesis . Cancer On the other hand, disruption of tissue innervation might cause accelerated tumor growth and metastasis , 96–101.Tumor innervation influences metastasis as the ingrown nerve endings release neurotransmitters , which enhance metastatic spread . To date2 adrenergic receptor/protein kinase A/BCL2-associated death protein anti-apoptotic signaling pathway [2-adrenergic receptors increases tumor growth rate and the number of metastasis [The increased expression of β-adrenergic receptor for catecholamines is associated with poor prognosis in breast cancer . Stress  pathway . Hence, tastasis . In conttastasis .The role of dopamine in cancer metastasis is not clear. Low levels of dopamine have been reported in stressed mice with ovarian carcinoma . In contA) and metabotropic (GABAB) receptors [A receptor. For example, HCC cell lines and human adjacent non-tumor liver tissues, express GABAA receptor. GABA inhibits HCC cell migration through the activation of GABAA receptor [A receptor enhances metastasis. The activation of GABAA receptors upregulates brain metastasis of breast cancer patients [A receptor subunit, Gabra3, which is normally not present in breast epithelial cells, is increased in human metastatic breast cancer which correlated with poorer patients survival [A receptor depends on the activated downstream molecules and signalling pathways. Murine (4 T1) and human (MCF7) breast cancer cell lines and human breast cancer tissues express GABAB receptor [B receptor mediates 4 T1 cell invasion and pulmonary metastasis via ERK1/2 signaling [B activation inhibits migration of PLC/PRF/5 and Huh 7 malignant hepatocyte cell lines in vitro [Plays a role in cancer metastasis via activation of ionotropic  signalling pathway [Plays a functional role in cellular proliferation, differentiation and apoptosis. In HCC, the release of ACh acting on androgen receptor promotes SNU-449 cell invasion and migration via activation of AKT and signal transducer and activator of transcription 3 (STAT3) signaling pathways . Nicotin pathway . ACh pro pathway . In addi pathway . M3R act pathway . In huma pathway , 139.Expression of SP is shown to exert functional effects on small cell lung cancer , pancreaMetastasis continues to be the main cause of cancer-related death. Although genetic compartments that influence metastasis have been identified, there are still needs to conduct comprehensive evaluation of the factors that contribute to cancer metastasis. This review demonstrates that the nervous system influences cancer metastasis through the release of neurotransmitters and neuropeptides leading to metastasis. However, sensory nerve fibres have been given less attention. Sensory stimuli activate pain transmission pathways which result in acute or chronic pain depending on the intensity and the nature of the stimulus , 153. CaIn conclusion, cancer cells can transduce neurotransmitter-mediated intracellular signaling pathways which lead to their activation, growth and metastasis. The findings reported here are primarily done in cancer cell lines and animal models. Therefore, better understanding the interaction between these signaling molecules and tumor cells in human cancers would enhance our knowledge on pathways promoting cancer metastasis.


Cardiomyopathy is a disease of the heart muscle leading to abnormal structure or function in the absence of coronary artery disease, hypertension, or valvular or congenital heart disease. Currently, cardiomyopathy is the leading diagnosis of heart transplant patients worldwide. Incorporation of next-generation sequencing strategies will likely revolutionize genetic testing in cardiomyopathy. The use of patient-specific pluripotent stem cell-derived cardiomyocytes for disease modeling and therapeutic testing has opened a new avenue for precision medicine in cardiomyopathy. Stem cell therapy, gene therapy, interfering RNA, and small molecules are actively being evaluated in clinical trials. Morphological subtypes include dilated cardiomyopathy, hypertrophic cardiomyopathy (HCM), arrhythmogenic right ventricular (RV) cardiomyopathy, left ventricular (LV) non-compaction, and restrictive cardiomyopathy. The global number of deaths and disability attributed to cardiomyopathy and myocarditis has steadily increased3. Cardiomyopathy represents the leading cause of cardiac transplantation4. The hereditary nature of most cardiomyopathies creates an opportunity for early detection through family screening but also has the potential for misdiagnosis5. How we classify, detect, and treat cardiomyopathy in 2017 and what is on the horizon are the focus of our review. In addition, infiltrative myocardial disease as an important differential diagnosis will be discussed when appropriate.Cardiomyopathy is a disease of the heart muscle leading to abnormal structure or function in the absence of coronary artery disease, hypertension, or valvular or congenital heart disease6. In that document, the following morphological and functional subtypes were recognized:As stated in the “Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of Cardiomyopathies”, a classification is an attempt to “bridge the gap between ignorance and knowledge”Dilated cardiomyopathy: characterized by ventricular dilatation and impaired contractionHCM: characterized by LV and/or RV hypertrophy, which is usually asymmetric and involves the interventricular septumRestrictive cardiomyopathy: characterized by restrictive filling and reduced diastolic volume of either or both ventricles with normal or near-normal systolic function and wall thicknessArrhythmogenic RV cardiomyopathy: progressive fibro-fatty replacement of RV myocardium, initially with typical regional and later global right and some LV involvement, with relative sparing of the septumUnclassified cardiomyopathies: unclassified cardiomyopathies include a few cases that do not fit readily into any group7. Primary cardiomyopathies are subclassified into genetic, mixed (genetic and non-genetic), and acquired. In 2007, the European Society of Cardiology proposed a new classification in which each morpho-functional subtype was subclassified according to familial/genetic and non-familial/non-genetic forms. The familial/genetic forms were subdivided into unidentified gene defect and specific disease subtype, and the non-familial/non-genetic into idiopathic and specific disease subtype8. “Specific cardiomyopathies” is used to describe cardiomyopathies associated with specific cardiac or systemic disorders. For example, a recent AHA document reviews diagnostic and treatment strategies for specific cardiomyopathies such as cardiac amyloidosis, cardiotoxins, peripartum cardiomyopathy, cardiac sarcoidosis, myocarditis, autoimmune cardiomyopathy, endocrine and metabolic cardiomyopathies, and genetic cardiomyopathies9. Nevertheless, the application of morphological criteria can be misleading because diseases with similar imaging findings can have completely different pathophysiological mechanisms 10.The 2006 American Heart Association (AHA) classification divided cardiomyopathies into primary, which are solely or predominantly confined to heart muscle, and secondary cardiomyopathies, which show pathological myocardial involvement as part of generalized systemic (multi-organ) disorders1. The use of a web-based application facilitates the implementation of this classification system (http://moges.biomeris.com/moges.html) (The MOGE(S) classification of cardiomyopathies proposes a descriptive phenotype and genotype nosology system that incorporates the following five attributes: morpho-functional phenotype, organ(s) involvement, genetic inheritance pattern, etiological annotation including genetic defect or underlying disease/substrate, and functional status of the patient and disease process using both the American College of Cardiology/AHA stage and New York Heart Association functional class. The MOGE(S) nosology system is the most recently proposeds.html) .12. Quartaet al. recently proposed a framework to navigate this uncertainty13. Their major criteria to diagnose cardiomyopathy include “1- Marked morphological abnormalities, clearly outside the limits of physiological remodeling, 2- Clear evidence of global or regional LV (and/or RV) systolic or diastolic dysfunction, or dynamic LV outflow obstruction at rest or on effort, 3- Frequent  and repetitive ventricular ectopic beats, which do not subside or tend to increase with exercise and/or are polymorphic, or runs of sustained or nonsustained ventricular tachycardia, 4- Symptoms such as dyspnea, angina, pre-syncopal or syncopal episodes of non-vasovagal nature , or reduced performance as assessed by cardiorespiratory testing, 5- A positive genetic test — when available and robust — or specific family history of cardiomyopathy and/or juvenile heart failure or sudden death; if unknown or not investigated, judgment should be suspended until relatives have been screened”.Because the presence of a morphological abnormality is not synonymous with cardiomyopathy , and the risk of developing genotype-positive and phenotype-negative cardiomyopathy is unknown, the clinician faces major challenges when evaluating an individual patientEchocardiography is often the first diagnostic modality used when cardiomyopathy is suspected. Although morphological two-dimensional echocardiography and ejection fraction are key elements for phenotype characterization, advanced echocardiographic modalities such as tissue Doppler and strain have made possible the detection of early stages of myocardial dysfunction.15. Machine learning models that incorporate longitudinal strain, among other volumetric and mechanical function variables, may help in the distinction between physiologic and pathologic hypertrophy16. In patients with sarcoidosis but without evidence or history of cardiac involvement, impairment of global LV longitudinal strain was associated with increased risk of cardiac events17. In patients exposed to anthracyclines, a relative percentage reduction in global longitudinal strain of at least 15% early during the course of therapy predicts the development of cardiotoxicity18. Abnormal regional patterns of strain are useful for the identification of specific cardiomyopathies such as apical sparring usually seen in patients with preserved ejection fraction heart failure due to cardiac amyloidosis or apical compromise in patients with apical HCM20. In patients with obstructive HCM refractory to medical therapy who underwent septal myectomy, basal septal (myocardium removed during myectomy) mean systolic strain and diastolic strain rate correlated within vitro measured myocardial contractile performance21. Longitudinal strain and early diastolic strain pre-transplant showed significant correlations with mRNA expression of titin isoforms, sarcoplasmic reticulum Ca2+ ATPase, and phosphorylated phospholamban in dilated cardiomyopathy22.Abnormal tissue Doppler signals may help to identify the presence of HCM in the pre-hypertrophic stage and to differentiate athlete’s heart from HCM23. Also, vortex formation has been correlated with functional capacity in a pilot study24.Vector flow mapping, a novel method of Doppler signal analysis, has shown that the cause of systolic anterior motion of the mitral valve and obstruction in HCM is early systolic ejection flow or isovolumetric vortical flow impacting the posterior aspect of the mitral valve and not Venturi forces related to high flows in the LV outflow tract26. Fractal dimension, a parameter used to quantify myocardial muscle trabecular complexity, is increased in HCM mutation carriers without LV hypertrophy27. LV structural abnormalities in patients with arrhythmogenic RV dysplasia have also been described with increased frequency29.Cardiac magnetic resonance imaging has become an integral part of cardiomyopathy evaluation and risk stratification. Apical–basal bundles and mitral valve abnormalities  have emerged as new phenotypic markers of HCM that are observed also in the pre-hypertrophic stage30. In HCM, for every 10% increase in LGE, there was a 40% increase in SCD events. Of particular importance is that, in patients who were considered to be at low risk of SCD by traditional risk stratification systems, the presence of LGE of at least 15% of the myocardial mass had a five-year risk of SCD of 6%31. In patients with Duchenne muscular dystrophy, the presence of LGE is associated with a progressive decline in LV ejection fraction (LVEF), and the magnitude of this decline is proportional to the number of myocardial segments with LGE32. In patients with cardiac amyloidosis, cardiac magnetic resonance imaging shows a characteristic pattern of global subendocardial LGE; in addition, patients with systemic AL amyloidosis show markedly increased non-contrast T1 relaxation times in the myocardium when compared with healthy controls and patients with aortic stenosis34.The presence, distribution, and burden of macroscopic myocardial scar tissue as measured by late gadolinium enhancement (LGE) are of particular importance. In a meta-analysis of 2,390 patients with various types of dilated cardiomyopathies, LGE was independently associated with higher risk of sudden cardiac death (SCD) and ventricular arrhythmias35. Increased interstitial fibrosis is a marker of subclinical cardiac involvement in patients who are carriers of lamin A/C gene mutation36.Extracellular volume fraction, which includes both diffuse and macroscopic patchy fibrosis using the modified look–locker inversion (MOLLI) recovery method, has been shown to correlate better with regional LV myocardial velocities than ejection fraction in patients with non-ischemic cardiomyopathyin vivo diffusion tensor cardiac magnetic resonance has been shown to characterize the microstructural dynamics by analyzing sheetlet (laminar microstructures 5–10 cardiomyocyte thick) mobility and orientation during the cardiac cycle. Reduced mobility is seen in both dilated cardiomyopathy and HCM. Nevertheless, in HCM, abnormal diastolic conformation is present, whereas abnormal systolic conformation characterizes dilated cardiomyopathy37.Recently,38.The use of four-dimensional (4D) flow (3D + time = 4D) allows the generation of 3D streamlines that permit the analysis of different flow patterns. The application of this technique in patients with HCM, with and without obstruction, revealed abnormal flow patterns in the ascending aorta in both groups. The significance of these findings is unknown39.99mTechnetium phosphate derivatives can bind to transthyretin in the myocardium and can be used to identify wild-type and mutant transthyretin-related amyloidosis. If amyloidosis is suspected and a scan is negative, this favors the diagnosis of light-chain amyloidosis40.Cardiac positron emission tomography evaluates the presence of perfusion and metabolic abnormalities. In cardiac sarcoidosis, the coexistence of myocardial perfusion defects and increased focal metabolic activity, which reflects active disease, is associated with increased risk of death and ventricular tachycardia41.Invasive hemodynamic evaluation is particularly useful to differentiate restriction from constriction. In this case, simultaneous pressure measurements of the right and left ventricles are performed ideally with high-fidelity conductance catheters to evaluate for the presence of enhanced ventricular interaction and systolic area index in the case of constrictive pericarditis. Careful evaluation is necessary to avoid missing the diagnosis of a potentially reversible condition42. The importance of tissue-based diagnosis for accurate prognostication in patients with unexplained cardiomyopathy has been known for a long time43. The procedure is usually performed by using fluoroscopic guidance, with increasing interest in the use of 3D echocardiography to decrease the chances of RV free wall biopsy44. The endomyocardial biopsy sensitivity can reach almost 100% in amyloidosis or approximately 30% in focal diseases such as sarcoidosis46. Usually, RV biopsy is performed, but the biventricular approach has been reported to increase diagnostic yield with a similar major complication rate in very experienced operators47. The performance of voltage mapping to guide endomyocardial biopsy has been described to increase diagnostic yield in patients with focal myocardial diseases such as sarcoidosis or lymphocytic myocarditis48.Transvenous endomyocardial biopsy is an invasive procedure with a reported major complication rate  of less than 1%50. Interestingly, the presence of focal derangement or diffuse lysis of myofilaments observed by electron microscopy in the endomyocardial samples obtained from the posterolateral LV wall of patients with dilated cardiomyopathy admitted with acutely decompensated heart failure was a strong predictor of death or heart failure re-hospitalization during a follow-up period of 4.9 ± 3.9 years51.In myocarditis, the use of immunohistochemistry to characterize inflammatory mechanisms, molecular techniques such as polymerase chain reaction to detect viral genomes, and—more recently—the analysis of non-coding transcripts such as microRNA hold the promise to continue to improve prognostication and treatment in this potentially lethal condition53. In cardiac sarcoidosis, the use of programmed electrical stimulation may assist in SCD risk stratification54. Approximately 30% of patients will fail to respond to cardiac resynchronization therapy, and many causes or associated factors have been identified 55. A novel mechanism to potentially improve cardiac resynchronization response is pacemaker-induced transient asynchrony. Six hours of daily RV pacing halted LV dilatation and myocyte dysfunction in a tachycardia-induced cardiomyopathy model in dogs56. The recognition of a significant premature ventricular contraction burden (>24%) should increase the suspicion of premature ventricular contraction-induced cardiomyopathy; this is important because ablation of the ectopic foci may lead to improvement of cardiac function57.Implantable cardioverter defibrillators are indicated for the prevention of SCD in selected patients with heart failure and reduced ejection fraction and HCM at high risk of sudden death58. The use of electro-anatomical mapping to guide endomyocardial biopsy has been previously described48.Electro-anatomic scar patterns differentiate RV outflow tachycardia from arrhythmogenic RV/dysplasia (ARVD) or cardiomyopathy. In ARVD, dominant sub-tricuspid scars with extensions toward the apex and RV outflow tract (RVOT) are seen where, in RVOT, isolated RVOT scar may be seen59.Most cardiomyopathies are monogenic disorders. Unexplained cardiomyopathy, family history of SCD or cardiomyopathy, and electrocardiogram suggesting an inherited arrhythmia should trigger genetic evaluationhttps://familyhistory.hhs.gov/FHH/html/index.html) if possible. Early consultation with genetics professionals is encouraged. It is important to convey the reason for consultation and potential implications to the patient. In cardiomyopathy, the main roles of genetic testing are to aid in the identification of family members at risk for the condition and to inform screening strategies.The first fundamental step is a family history using a standardized template  and controls (n = 249), respectivelyet al. have reported that patients with HCM who have a mutation affecting the thin filament are at increased risk of developing LV dysfunction, heart failure, and severe diastolic dysfunction when compared with patients with thick filament mutations61. Nevertheless, it is important to recognize that HCM is a disease where gene-phenotype correlations are more notable for their absence than presence.Coppini62. In addition, in a large cohort of patients with ARVD/C who had pathogenic mutations, desmoplakin mutations were overrepresented in patients with SCD/ventricular fibrillation as a presenting symptom63. In the same study, the negative prognostic implications of having more than one pathogenic mutation and male sex were also reported.Studies showing the impact of genotype in prognosis in ARVD/C have been reported. Patients with ARVD/C and carriers of a desmosomal gene mutation have worse outcomes when compared with those with titin mutations64. Pan-cardiomyopathy panels have not been shown to increase the diagnostic sensitivity in HCM65. The decrease in cost and turn-over time of next-generation sequencing strategies, including exome (sequences the genes involved in protein synthesis) and whole genome (sequences both coding and non-coding), has led some institutions to adopt this strategy. From a clinical standpoint, one of the main challenges of non-directed second-generation sequencing is the increase in the identification of variants of unknown significance; this is non-actionable genetic information66. Nevertheless, recent reports combining these techniques with functional studies show encouraging results in detecting new pathogenic mutations67. The creation of large repositories of genetic and phenotypic information, along with powerful analytic techniques, will shape the implementation of next-generation sequencing68.Future applications may include the development of genotype-specific therapies, and pilot studies of pharmacologic therapies in the pre-phenotype phase have been completed (see below). If concomitant congenital malformations are present and a chromosomal disorder is suspected, a chromosomal microarray analysis should be considered. When cardiomyopathy is the main feature and genetic abnormalities at the nucleotide level are suspected, several options are available. Gene panels directed to specific morpho-functional phenotypes that screen for pathogenic mutations are available. Results of genetic testing have five categories: pathogenic, likely pathogenic, variables of unknown significance, likely benign, and benign. The diagnostic yield of these panels is variable and ranges from 50 to 60% in ARVD and from 20 to 30% in dilated cardiomyopathy69.The incorporation of genetic information in epidemiologic studies with long-term follow-up has opened a new chapter in understanding the natural history of disease. Amyloidosis is a systemic disease due to the deposition of misfolded proteins that may affect the heart. There are two main forms of cardiac amyloid disease: AL amyloidosis due to immunoglobulin light chains and transthyretin amyloidosis. Patients with transthyretin may have wild-type  or genetic variants. The V122I variant, where isoleucine is substituted for valine, has a prevalence of approximately 3% in African-Americans and is the most common point mutation associated with hereditary transthyretin amyloidosis. Carriers of this mutation were believed to have an increased risk of death. In a study that included 3,856 African-Americans, 124 carriers were detected, and over a follow-up period of 21.5 years, those patients were at increased risk of heart failure development, but there were no statistically significant differences in mortality70.Other genetic variants include Val30Met  and Thr60Ala (Irish), both of which commonly affect the nerves and the heart. Nevertheless, early cardiac involvement is not as frequent in Val30Met71. In this regard, the development of transcription factor-mediated reprogramming techniques, which allowed the production of human-induced pluripotent stem cells (hiPSCs), represents a major step forward72. Protocols for the production of patient-specific pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are available73. Applications of hiPSC-CMs include disease modeling, regenerative medicine, drug discovery, and toxicity screening74. For hiPSC-CMs that carry a mutation in cardiac transcription, factor TBX20 develops phenotypic characteristics of LV non-compaction cardiomyopathy. In this experiment, abnormal transforming growth factor-beta (TGF-β) signaling was detected. Inhibition of TGF-β signaling and genomic correction of the TBX20 mutation were sufficient to reverse the phenotype75. Using patient-derived hiPSC-CMs, the mechanisms by which titin mutations cause sarcomere insufficiency in dilated cardiomyopathy were explored76. In another exciting experiment, hiPSC-CMs from patients who experience doxorubicin cardiac toxicity were more sensitive toin vitro doxorubicin toxicity than hiPSC-CMs of patients who did not experience doxorubicin toxicity. Several mechanisms were identified, including decreased cell viability, impaired mitochondrial function, metabolic derangements, and increased production of reactive oxygen species77. This finding suggests that hiPSC-CMs could be a potential tool to predict chemotherapy-induced cardiotoxicity.Precision medicine is an emerging approach for disease treatment and prevention that takes into account individual variability  and is of particular relevance for cardiomyopathy78. One of the advantages of detecting genotype-positive phenotype-negative patients is the potential to intervene and alter the trajectory of disease. In a pilot and first-of-its-kind study, 38 patient carriers of pathogenic sarcomere mutations associated with HCM, but without LV hypertrophy, were randomly assigned to diltiazem or placebo. Patients who received diltiazem showed stable LV diameter and mean thickness-to-dimension ratio compared with control patients who showed a decrease and an increase in LV diameter and mean thickness-to-dimension ratio, respectively79.Mutation silencing therapy in a murine model of HCM has shown that irreversible triggers to phenotypic expression occur early in development (<6 weeks of life)81.There has been pre-clinical evidence of the potential benefit of renin-angiotensin axis inhibition in modifying the course of HCM in a randomized controlled trial that included 133 patients with obstructive and non-obstructive cardiomyopathy, but the administration of losartan, though well tolerated, did not show significant difference when compared with placebo with regard to the primary endpoint (change in LV mass as measured by computed tomography or cardiac magnetic resonance imaging)83.The presence of LGE has been used to identify patients with Duchenne and Becker muscular dystrophies who have normal LVEF but who are at higher risk of developing cardiac dysfunction. In this particular patient population, treatment with eplerenone (Duchenne) and angiotensin-converting enzyme inhibitor (Duchenne and Becker) delayed and attenuated adverse cardiac remodeling84. In non-ischemic dilated cardiomyopathy, myocardial blood flow at rest and after cold pressor test improved after 12 weeks of cardiac rehabilitation85.The value of moderate exercise training in selected patients with HCM has been recently evaluated in a pilot randomized trial. After 16 weeks, patients randomly assigned to the unsupervised exercise protocol had a small but significant increase in exercise capacity as measured by cardiopulmonary exercise testing with no significant adverse events86. The intravenous administration of MYK-461 decreased contractility and eliminated systolic anterior motion of the mitral valve, relieving outflow obstruction in a feline model of HCM87. Initial experience in humans shows encouraging results with a dose-dependent reduction of contractility and abolition of LV outflow gradients in two patients. Of note, one patient experienced asystole after receiving the highest dose studied, but it resolved without intervention88.MYK-461, a small molecule which binds to myosin decreasing its ATPase activity in a dose-dependent manner and consequently sarcomere contractility, has been shown to prevent the development of the HCM phenotype in a murine model of HCM if administered early (8–15 weeks of age) in the pre-hypertrophic stage, and it partially reversed structural abnormalities if administered once the hypertrophic phenotype became manifestet al. described that, in a murine model of Noonan syndrome and Noonan syndrome with lentigines, the administration of low-dose dasatinib (tyrosine kinase inhibitor) improves cardiomyocyte contractility, reduces fibrosis, and—if administeredin utero—rescues HCM phenotype89.Noonan syndrome and Noonan syndrome with multiple lentigines (formerly LEOPARD syndrome) as other RASopathies are autosomal dominant disorders caused by germline missense mutations of the Ras/mitogen-activated protein kinase (Ras/MAPK) signaling pathway; one of the phenotypic abnormalities is the development of HCM. A hyper-tyrosil phosphorylated form of protein-zero related (PZR) is present in the hearts of mice with those conditions. YiLMNA gene encodes lamins C and A that are the major constituents of nuclear lamina, a proteinaceous meshwork that gives structural support to the nucleus and enables correct gene expression and DNA repair. Lamins A and C are generated through alternative splicing90. Laminopathies are a diverse group of disorders caused byLMNA gene mutations and the most common one is dilated cardiomyopathy91. Current pathophysiological mechanisms ofLMNA mutations include increased susceptibility to mechanical stress, altered gene expression, and accumulation of pre-lamin A93. The feasibility of alternative splicing modulation with antisense oligonucleotides to increase lamin C and decrease pre-lamin A accumulation has been successfully tested in fibroblasts of patients with Hutchinson-Gilford progeria syndrome and a mouse model94. The recognition of abnormal activation of the MAPK and Akt/mTOR  pathways has led to promising experimental data using protein kinase inhibitors and mTOR inhibitors, respectively, to block the development of cardiac dysfunction in mouse models of laminopathies96.The97.Constitutive activation of mammalian target of rapamycin complex 1 (MTOR-1) by tuberous sclerosis mutations is a recognized mechanism of tumor formation in this syndrome. In a patient with tuberous sclerosis and cardiomyopathy in which phosphorylation of ribosomal protein S6 (marker of MTOR-1 activation) was detected in endomyocardial biopsy, the administration of everolimus (MTOR inhibitor) led to improvement in systolic function and LV dimensions98. Increasing cardiac contractility by targeting molecules related to calcium cycling  or increasing beta-adrenergic system function  and enhancing stem cell tissue repair by increasing the expression of stem cell-derived factor 1 (SDF-1) have undergone clinical trial evaluation99. Calcium upregulation by percutaneous administration of gene therapy in patients with cardiac disease (CUPID 2) that included 250 patients with NYHA (New York Heart Association) functional class II–IV and LVEF of not more than 35% who were randomly assigned to intracoronary infusion of adeno-associated virus loaded with SERCA gene versus placebo failed to achieve the primary endpoint of recurrent events  with a median follow-up of 17.5 months. Nevertheless, the absence of significant adverse events is promising and has helped to enhance the interest in this therapeutic strategy100. A recent phase II trial has shown a significant increase in LVEF in patients who received intracoronary delivery of adenovirus hAC6 when compared with placebo101.Increased understanding of the molecular mechanisms of heart failure have led to the recognition of potential targets for gene therapy102. In a previous article, trans-endocardial injection of allogenic mesenchymal stem cells was superior to auto-mesenchymal stem cells in terms of efficacy  and safety with an extremely low risk of allosensitization103. A phase I clinical trial of transplantation of scaffold-free cell sheets derived from autologous muscle to the epicardial surface through left thoracotomy in patients with severe LV dysfunction due to ischemic (n = 15) or dilated cardiomyopathy (n = 12) has shown promising results regarding safety with no major procedural adverse events and efficacy with improvement in symptoms, 6-minute walk test, and LVEF104.Intravenous allogenic mesenchymal stem cells were shown to be safe and effective in improving the 6-minute walk test and quality-of-life measures in patients with non-ischemic cardiomyopathy with a mean LVEF of 31%. There was no evidence of myocardial scar as measured by LGE in patients on maximal medical therapy70. In light-chain amyloidosis, the use of an amyloid fibril-reactive chimeric monoclonal antibody is undergoing clinical trial evaluation. This approach was shown to be safe in a phase I clinical trial that included six patients with refractory amyloidosis. Those three patients showed organ response: two cardiac and one gastrointestinal105.Novel therapies that block abnormal protein synthesis such as small interfering RNA and antisense oligonucleotides are undergoing phase III clinical trial evaluation in transthyretin amyloidosis, the most common form of cardiac amyloidosis106. Anti- tumor necrosis factor therapy has been shown to be ineffective and even potentially harmful in this patient population in large randomized clinical trials107. Non-specific immunomodulation was also unsuccessful in reducing death from any cause and hospitalization from cardiovascular causes108. There is increasing interest in the manipulation of the innate immune system109. Toll-like receptors, which are the primary receptors of the innate immune system and related molecules , constitute attractive targets that are the subject of intense research111. In addition, the recognition that embryonic-derived macrophages that have anti-inflammatory properties and promote tissue regeneration are replaced in heart failure with monocyte-derived macrophages that have pro-inflammatory properties may open a new pathway to cardiac recovery112.Inflammation is a key player in the development and progression of heart failure113. A novel operative technique that consists of trans-aortic cutting of thickened secondary mitral valve chordae seems to be effective in relieving outflow tract obstruction in patients with HCM and mild septal thickness with the advantage of avoiding additional mitral valve procedures114. This technique is based on the pathophysiological hypothesis that fibrotic and retracted secondary chordae may cause abnormal tethering of the anterior mitral valve and favor the displacement of the “slack portions of the leaflet (and attached primary chordae) into the LV outflow tract”114.In HCM, surgical septal myectomy often resolves mitral regurgitation related to systolic anterior motion of the mitral valve; nevertheless, some patients need additional mitral valve procedures such as mitral valve repair or replacementThe number of patients affected by cardiomyopathies is increasing. Given the strong genetic basis of many cardiomyopathies, a complete family history is mandatory. Judicious use of cardiac imaging is extremely useful in defining the morpho-functional phenotype, informing prognosis, and detecting subclinical disease. Genetic testing is being increasingly incorporated into clinical practice. MOGES classification provides a good framework to facilitate communication and patient classification. The use of patient-specific pluripotent stem cell-derived cardiomyocytes for disease modeling and therapeutic testing is exciting and hopefully will be incorporated into clinical practice in the near future. Gene therapy, small molecules, small interfering RNA, and antisense oligonucleotides are being tested in clinical trials.


The hedgehog signaling cascade is an evolutionarily conserved pathway that regulates multiple aspects of embryonic development and plays a decisive role in tissue homeostasis. As the best studied member of three hedgehog ligands, sonic hedgehog (Shh) is known to be associated with kidney development and tissue repair after various insults. Recent studies uncover an intrinsic link between dysregulated Shh signaling and renal fibrogenesis. In various types of chronic kidney disease (CKD), Shh is upregulated specifically in renal tubular epithelium but targets interstitial fibroblasts, thereby mediating a dynamic epithelial-mesenchymal communication (EMC). Tubule-derived Shh acts as a growth factor for interstitial fibroblasts and controls a hierarchy of fibrosis-related genes, which lead to the excessive deposition of extracellular matrix in renal interstitium. In this review, we recapitulate the principle of Shh signaling, its activation and regulation in a variety of kidney diseases. We also discuss the potential mechanisms by which Shh promotes renal fibrosis and assess the efficacy of blocking this signaling in preclinical settings. Continuing these lines of investigations will provide novel opportunities for designing effective therapies to improve CKD prognosis in patients. It is estimated that the prevalence of chronic kidney disease (CKD) is about 10%–12% of the adult population worldwide . As a geDrosophila in 1980, and the vertebrate hedgehog proteins were discovered in 1993 . Hhip and Smo colocalize at the cell surface, Hhip has no effect on activity of Smo but modulates Smo localization. In addition, Hhip1 also sequesters hedgehog ligands  after kidney injury , desmin, Snail1, fibronectin, and collagen I expression , suggesting that Shh not only is sufficient but also necessary for fibrosis development in the kidney. This conclusion is also supported by genetic and pharmacologic manipulations of the Shh downstream mediators Smo or Gli proteins. It is shown that the kidneys in Gli1-deficient mice are protected against the development of tubulointerstitial fibrosis after UUO . Smo inhlacZ mutant mice, the so-called hedgehog-reporter mice, which harbor a β-galactosidase  “knock-in” mutation. Under the control of the native Gli1 upstream promoter/enhancer elements, lacZ expression in these mice authentically recapitulates the expression pattern of the endogenous Gli1 mRNA. Results show that interstitial fibroblasts, including platelet-derived growth factor receptor (PDGFR)-β-positive pericytes, are identified as principal Shh-responding cells in vivo in UUO and IRI models  of the G-protein-coupled receptor (GPCR) superfamily . Recentlin vitro, and it reduces matrix expression and mitigates fibrotic lesions in vivo after UUO and IRI  has been utilized in the treatment of experimental renal fibrosis . CPN is  and IRI . Meanwhi and IRI . However and IRI . Therefofibrosis . These dThe discovery of CPN and its derivative has heralded extensive research for development of Smo antagonist. A major breakthrough is the recent approval of a novel Smo antagonist Erivedge/Vismodegib (GDC-0449) in the Shh field by the FDA for treating metastatic BCC and locally advanced BCC deemed untreatable by surgery or radiation . Howeverin vivo  communicates with mesangial cells? What is the extent of interaction between Shh and other pathologic signal pathways? It will be of great interest to see these different aspects of Shh signaling unveiled in the near future.


Sensing of microbe-associated molecular patterns or danger signals by innate immune receptors drives a complex exchange of information. Innate receptor signaling not only triggers transcriptional events but also induces profound changes in metabolic fluxes, redox balance, and metabolite abundance thereby influencing immune cell function. Mitochondria are at the core of metabolic adaptation to the changing environment. The close interaction between mitochondrial metabolism and immune signaling has emerged as a central regulator of innate sensing. Metabolic processes generate a constant flow of electrons that eventually end up in the mitochondrial electron transport chain (ETC). Two electron carriers and four respiratory complexes that can assemble as larger molecular supercomplexes compose the ETC in the mitochondrial inner membrane. While the meaning and biological relevance of such structural organization is a matter of passionate debates, recent data support that innate stimuli remodel the ETC. We will review the function of mitochondrial metabolism and ETC dynamics as innate rheostats that regulate signaling, transcription, and epigenetics to orchestrate innate immune responses. Ligation of pattern recognition receptors, cytokine receptors, and phagocytosis of dying or dead cells provoke key changes in myeloid cell metabolism that are only beginning to be explored . As an e2), whose relative proportion depends on the nature of the fuel used . Many catabolic processes indeed supply electrons to the ETC in the form of reducing equivalents of nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FADHuel used . The capuel used . Mitochouel used . Mitochoc] and four respiratory complexes [complexes I–IV (CI–CIV)], which, excluding CII, can assemble as larger molecular supercomplexes (SCs) in the mitochondrial inner membrane  to generate ATP. Recent results support the notion that innate sensing of microbial features leads to adaptations in SC assembly and electron flow through the ETC in mouse macrophages (The ETC comprises two electron carriers [coenzyme Q (CoQ)/ubiquinone and cytochrome membrane   that binds to mitochondrial-associated membranes connecting the endoplasmic reticulum to the outer mitochondrial membrane . RIG-I bSalmonella typhimurium  cycle and maintain global metabolic flux , 8, 47 lHow innate immune receptors regulate lipid metabolism in macrophages is a field of active research. On the one hand, engagement of TLRs upon recognition of various bacteria enhances fatty acid uptake and incorporation to triglycerides for storage and simultaneously decrease fatty acid oxidation and lipolysis suggesting that lipids may serve other needs than energy production in activated mouse macrophages , 49, 50.2. The intramitochondrial NADH/FADH2 ratio depends on the nature of the fuels that feed the mitochondrial metabolism and the respiratory chain adapts to these fuel source fluctuations , promotes a distinct metabolic reprogramming compared to pro-inflammatory M1) mouse macrophage stimulating LPS/IFN-γ indicates that mitochondrial metabolism adjusts to the type of immune responses required to eliminate the threats encountered  mouse ma. M2 mous2, and this function as electron carrier drives modulation of ROS signaling  animals. Consistently, UCP2-deficient macrophages generated more ROS in response to T. gondii underlying this enhanced parasite-killing capacity  is highly expressed in mouse and human macrophages during inflammation. It metabolizes rial ROS . Importarial ROS  (Figure rial ROS . IFNs in killing . In addimulation . Reorganmulation  (Figure mulation . Notablymulation .Since mitochondria have emerged as key conductors of the metabolic adaptations to the innate sensing by modulating the electron flow in the ETC , 19, a kDS, ME, and JG conceived and wrote the manuscript. JG did the figures.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.


Recurrent hepatocellular carcinoma (HCC) in the extrahepatic bile duct is rare with most cases diagnosed after manifesting sudden obstructive jaundice. Here, we report an extremely rare case of recurrent HCC in the common bile duct due to hematogenous metastasis.A 66-year-old man underwent an extended left hepatectomy for HCC in the medial segment of the liver. Fifty months later, he presented with sudden obstructive jaundice. Endoscopic retrograde cholangiography showed a space-occupying lesion in the common bile duct, which was suspected as cholangiocarcinoma. Therefore, he underwent extrahepatic bile duct resection and choledochojejunostomy with lymph node dissection. Macroscopically, a polypoid tumor and several nodular tumors were found in the common bile duct, which was obstructed by a tumor thrombus. Histopathologically, the tumors were diagnosed as metastases from the HCC resected 50 months before. Several distinct, nodular tumors were observed in the subepithelium of the common bile duct and had invaded some blood vessels. These findings support the conclusion that the HCC metastasized hematogenously to the extrahepatic bile duct.Recurrent HCC in the extrahepatic bile duct due to hematogenous metastasis is rare, and it is difficult to diagnose. Further similar cases should be accumulated for clarifying the pathological mechanism. Curable hepatocellular carcinoma (HCC) is gradually increasing because of improved follow-up and imaging diagnosis protocols for chronic liver diseases. However, a therapeutic strategy for metastasis and recurrence of HCC remains a difficult topic. Recurrent HCC in the extrahepatic bile duct is somewhat rare, and almost all cases are diagnosed after the manifestation of sudden obstructive jaundice . We repoA 66-year-old man with hepatitis C underwent an extended left hepatectomy for HCC in the medial segment of the liver. Preoperative dynamic computed tomography (CT) and intraoperative evaluation revealed no lymph node metastasis Fig. . HistopaMacroscopically, an approximately 10-mm-diameter polypoid tumor continuous to the epithelium and a tumor thrombus were found in the dilated common bile duct, and several nodular tumors were observed subepithelially Fig. . The lesHCC in the major bile duct, especially in the common bile duct, can cause obstructive jaundice . Such HCThere are some rare reports of extrahepatic HCC without primary hepatic parenchymal lesions , 11; henHCC recurrence can be explained by the following points: (i) invasive recurrence from microscopic bile duct invasion; (ii) infiltrating recurrence of tumor thrombus having been incompletely removed during primary hepatectomy; (iii) invasion from the surrounding tissues of the common bile duct; or (iv) recurrence of hematogenous metastasis with microscopic venous invasion. In this case, no microscopic bile duct invasions were observed in the primary or recurrent HCCs. Furthermore, most recurrences originating from tumor thrombus have been reported to develop within a year of surgery , unlike In this case, because (a) there were severe portal invasion, (b) tumor thrombus was located in the left portal vein, and (c) there were several daughter nodules of microscopic intrahepatic metastasis, the patient had high chance of recurrence. Despite such high-risk histopathological findings, it is very interesting that he had a disease-free interval of 50 months before recurrence.The effectiveness of surgery for recurrence originating from tumor thrombus has been previously reported , 14. AltWe report a patient who developed an extremely rare recurrent HCC in the common bile duct due to hematogenous metastasis. Recurrent HCC in the extrahepatic bile duct is rare and presents a poor prognostic course. Further similar cases should be accumulated for clarifying the pathological mechanism.


Behavioral alterations emerging after central or peripheral vision loss suggest that cerebral reorganization occurs for both the afferented and deafferented early visual cortex (EVC). We explored the functional reorganization of the central and peripheral EVC following visual field defects specifically affecting central or peripheral vision. Compared to normally sighted, afferented central and peripheral EVC enhance their functional connectivity with areas involved in visual processing, whereas deafferented central and peripheral EVC increase their functional connectivity with more remote regions. The connectivity pattern of afferented EVC suggests adaptive changes that might enhance the visual processing capacity whereas the connectivity pattern of deafferented EVC may reflect the involvement of these regions in high-order mechanisms. Characterizing and understanding the plastic changes induced by these visual defects is essential for any attempt to develop efficient rehabilitation strategies. The macular and peripheral parts of the retina constitute an intricate functional unit. Their combined input is responsible for the impression of a homogenous image over the entire visual field. This functional unity is not present from birth since ontogenically, the central and peripheral parts of the retina mature sequentially. Unlike the periphery, the central retina is immature at birth and only develops completely years later12Adaptive strategies such as the eccentric fixation employed in the case of central visual field defects induce proportional functional changes in the peripheral early visual cortex (EVC)5101314131417181011121314101112Stargardt macular dystrophy (SMD) is a well-documented bilateral, inherited retinal disorder that induces well-circumscribed, central visual defects20222425We explored the changes induced by partial visual loss by analyzing resting-state functional connectivity (rs-FC), a method that places few demands on patients since they perform no task during scan acquisition. Resting-state fluctuations are well-organized into networks previously identified in a range of cognitive tasks27282930313233343537383939404138394444Twelve Stargardt macular dystrophy (SMD) subjects , presenting a central scotoma, 10–20 degrees in diameter , without foveal sparing, and best-corrected visual acuity equal or superior to 20/400 (measured by EDTRS charts). Ages ranged from 18 to 58 years .Twelve retinitis pigmentosa tunnel vision (RPTV) subjects , presenting a central residual visual field limited to a 10–20 degree diameter , and best-corrected visual acuity equal or superior to 20/40 (measured by EDTRS charts). Ages ranged from 18 to 62 years .Fourteen normally sighted controls , with normal routine ophthalmological examinations. Ages ranged from 18 to 59 years .Subjects were recruited and assigned to the three groups as follows:Subjects were matched for age, and no subject had any reported neurological or psychiatric diseases see . The EthfMRI was conducted with a whole-body 3T clinical imager (Sigma Horizon) using an eight-channel head coil. In each scanning sequence, 32 contiguous axial T2*-weighted gradient-echo echo-planar images  were recorded to encompass the entire brain. 180 volumes were acquired including 4 “dummy” volumes obtained at the start of the session. Scan duration was 9.25 minutes for the EPI sequence. Off-line, T2*-weighted images were co-registered and overlaid on the corresponding anatomic T1-weighted gradient-echo images . During the scan, subjects were supine in the MRI scanner and wore earplugs to compensate for the noisy environment. Subjects were instructed to keep their eyes closed. No explicit task was required.fMRI data were preprocessed using the BrainVoyager QX 2.8 software package  and complementary software written in MATLAB R2009a . Preprocessing of functional scans successively included: 3D motion correction , slice-time correction, band-pass filtering between 0.01 and 0.1 Hz, voxel-to-voxel linear regressionet al. 1999).External functional localizers were used to define the seed region-of-interest (ROI) from visual localizers  in the volume. Each of these localizers was extracted from a group of normally sighted controls and analyzed in normalized Talairach space using a random effect GLM which enabled generalization of the findings to the population  was scanned in a standard phase-encoded retinotopic mapping experiment, using ring (eccentricity mapping) and wedge (polar mapping) stimuli39For supplementary ROI analyses, we also used external functional localizers from previous studies by our team4042To validate the localizer definitions, we also ran a control analysis see  at the ie.g., the central EVC) served to extract and z-transform the blood-oxygen-level dependent time course of this region in volumee.g., the peripheral EVC) to suppress the shared variance and conserve only the unique variance corresponding to the ROI predictor.A partial correlation analysis39A random-effect analysis based on the general linear model (GLM)39i) the age at disease onset and, (ii) the duration of the visual deficit  for multiple comparisons) and the FC patterns found in the between-group maps .For each patient group, we also tested the correlations between the following variables  were performed.To summarize, an ROI-based functional connectivity analysis applied to the brain resting-state fMRI data was conducted on all groups. We used data from the seeded regions to establish resting-state functional connectivity maps intra-group. We then ran an ANOVA on all the subjects and post-hoc analysis to determine differences between groups in volume. The maps were then converted and displayed on the surface , enabling a complete view of the brain.All the results presented below were statistically significant .Afferented central EVC in the normally sighted exhibited a positive FC with the bilateral inferior and middle occipital and bilateral posterior part of the lingual gyri, bilateral orbitofrontal cortex but a negative FC with the bilateral inferior frontal gyrus, insula, cuneus, parieto-occipital sulcus, precuneus, cingulate sulcus, superior temporal and frontal gyrus, left intraparietal sulcus, inferior parietal lobule, precentral gyrus and right superior frontal sulcus , insula, right anterior cingulate gyrus and right cuneus, right intraparietal sulcus, precentral sulcus and the posterior part of superior, middle and inferior frontal gyri .Afferented peripheral EVC in the normally sighted exhibited positive FC with the bilateral cuneus, precuneus, posterior cingulate, lingual, parahippocampal, fusiform, superior and middle occipital gyri, posterior middle temporal gyrus and superior temporal sulcus, anterior superior temporal gyrus and right orbitofrontal and anterior cingulate cortex. It exhibited a negative FC with the bilateral inferior and middle frontal gyri, precentral sulcus, inferior frontal sulcus, inferior parietal lobule  and left superior frontal gyrus .As compared to normally sighted, the RPTV subjects showed increased FC of the afferented central EVC with the left middle occipital and left superior temporal gyri. There was also reduced central EVC FC with the anterior part of the right middle temporal gyrus, as well as the bilateral ventral anterior cingulate cortex see . SMD subA comparison of the two groups of visually deprived subjects indicated that the SMD subjects presented increased FC between the deafferented central EVC and the right cuneus and precuneus, the right inferior parietal lobule and the left superior parietal lobule see . RPTV suAs compared to normally sighted, the RPTV group exhibited increased FC with the right inferior parietal lobule, as well as with the posterior and anterior regions of the medial frontal gyrus see . In contComparing both groups of visually-deprived subjects showed increased FC of the SMD subjects’ still afferented, peripheral EVC with both ventral and dorsal stream regions – the bilateral middle occipital, inferior occipital gyri, the right superior occipital gyrus, the right lateral occipital cortex, the bilateral lingual and fusiform gyri as well as the right parahippocampal gyrus see . FC was Both S1 respective lip and foot areas were perfectly connected between hemispheres in the three groups see  and 6. TThe left foot area was functionally connected with bilateral post central gyrus, precentral gyrus, premotor cortex, and precuneus in all three groups. In all groups the left foot area also exhibited negative FC with bilateral IFG and in peripheral visual field defect (RPTV) and in normal vision also with the anterior insula as well. Moreover, the central visual field group (SMD) exhibited increased FC between the left foot area and the left SOG and the right inferior occipital gyrus whereas the peripheral visual field defect group (RPTV) exhibited increased FC with the right peripheral EVC, LOC, superior temporal gyrus and bilateral SPL see .In patients with a peripheral visual field defect compared to normal vision, the left S1 lip area showed increased FC with STG and decreased FC with an area that overlaps V5/hMT+ and EBA as well as SPL see . In patiWe found no FC difference for the left foot area between normally sighted and patients with peripheral visual field loss (RPTV) see . In patiThe main results were summarized in SMD and RPTV models were implemented to evaluate the effects of central and visual loss on the FC of the EVC. The five main findings can be summarized as follows: (1) in both patient groups, deafferented EVC exhibited a different FC pattern than the corresponding regions in normally sighted, suggesting that either the central or peripheral EVC could reorganize after deafferentation but more interestingly (2) we also found a different FC pattern in the patients’ EVC that still received the residual visual input, implying that such reorganization also impacts afferented visual areas; (3) we found more changes in FC to peripheral than to central EVC (in both afferented and deafferended states); (4) compared to normal vision, the patients’ afferented central or peripheral EVC demonstrated increased FC with many areas involved in visual processing whereas the deafferented central or peripheral EVC showed increased FC with remote regions; (5) The contrast between deafferented and isolated afferented early visual cortex revealed patterns of FC differences that were similar, but more complex compared to those observed in comparison to normally afferented visual cortex.In the normally sighted, the central and peripheral early visual cortex exhibited a positive FC with areas of the dorsal and ventral stream. The positive FC of the central and peripheral EVC was roughly similar to previous descriptions of the entire EVC connectivity with the exception of the primary sensorimotor cortexCompared to the subjects with normal vision, the afferented central EVC in RPTV exhibited increased FC with the left MOG and STG, whereas their deafferented EVC periphery showed decreased FC with these same regions. Interestingly, in normal vision these regions had stronger canonical connections with the peripheral EVC as found in previous studies see 575859. I5758606162Compared to the subjects with normal vision, the afferented peripheral EVC in SMD subjects showed an increased FC with areas of the ventral stream  and associated functional areas . Most of these regions are involved in the face perception network6464Compared to the normally sighted, the deafferented peripheral EVC in RPTV group exhibited increased FC with the IPL/IPS and MFG. Interestingly, the isolated afferented central EVC showed decreased FC with the same region. IPS/IPL plays a role in top-down control, attention, multisensory integration, visuomotor coordinate transformation and task-demand in normal subjects587071Compared to the normally afferented (sighted) and isolated afferented central EVC (RPTV), the deafferented central EVC of the SMD exhibited increased FC with the cuneus, the parieto-occipital sulcus (POS) and the precuneus. The participation of these regions in switching attention, orientation selectivity and visuomotor processingAt the intra-group level, both the deafferented central (SMD) and peripheral (RPTV) EVC showed a positive FC with the posterior thalamus/pulvinar . The pulvinar is involved in attentional processes and is thought to play a fundamental role in cortico-cortical communication1313The contrast between isolated afferented EVC and deafferented EVC is difficult to interpret as it compares two groups with a modified FC. It was primarily intended to be a control for the comparison to the normally afferented visual cortex. In fact, the differences in FC between the isolated afferented central EVC in the RPTV and the deafferented central EVC in the SMD group were roughly similar to the differences observed when compared to the normally afferented central EVC, with the exception of the MOG. The lack of a FC difference in MOG, consistent with the results mentioned above, might reflect the increased adaptive connectivity of both the deafferented and isolated afferented central EVC with this region. The isolated afferented central EVC of RPTV also exhibited a relative increase in FC with the IOG and fusiform gyrus, which are areas of the ventral processing stream.Compared to the deafferented peripheral EVC in the RPTV patients, the isolated afferented peripheral EVC in the SMD patients exhibited an increased FC with the right sensorimotor cortex, superior temporal gyrus and bilateral LOC. Thus, this unique contrast between afferented and deafferented peripheral EVC also exposes the multisensory connections of the afferented peripheral EVC with sensorimotor and auditory areas3757The intra-group ROI analyses based on somatosensory seeds validate our resting-state partial correlation approach by showing that the somatosensory topographic system could be mapped in our sighted population as well as in both groups of visually-impaired patients see  and 6. T40The analysis seeding the foot sensory projection area also yielded consistent results. The regression of the lip projection area resulted in a negative FC between the foot area and the inferior frontal gyrus and insula. In central visual field loss, the foot area is functionally connected to the isolated afferented peripheral EVC see . This coet al. found that resting-state correlations between V1 and V2/V3 were retinotopically organized in normally sighted, early blind as well as anophthalmic subjects suggesting that retinal waves or visual experience are not necessary to develop a resting-state retinotopic pattern787980To the best of our knowledge, few studies39nd hypothesis). An alternative explanation for the increased FC of the sensory deprived visual cortex is that it represents noise generated by aberrant autonomous activityth hypothesis). This scenario is unlikely because it would imply strengthened connections between the deafferented EVC and regions to which they are strongly linked in normal vision, an effect that was not confirmed by our data. Rather, the enhanced connectivity of the deafferented primary visual cortex concerned long-range connections involved in higher-order processing. However, as suggested by the thinning of the gray matter in the deafferented EVC in conditions inducing similar visual field defects8384858687Overall, the FC pattern of the part of the EVC receiving the remaining visual input supports the first hypothesis that the isolated afferented EVC strengthens the local connections, presumably to tune-up visual information processing. In the deafferented EVC, remodeling concerns more remote connections, and this pattern lends support to the third hypothesis that deafferented EVC likely diverts its processing capacity to higher-order functions that can optimize the residual visual function. The absence of increased local connectivity in the deafferented compared to the normally afferented EVC or its decreased local connectivity when compared to the isolated afferented EVC do not support the view that deafferented EVC rewires the same type of information as the afferented EVC .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Wolbachia are widespread heritable endosymbionts of arthropods notorious for their profound effects on host fitness as well as for providing protection against viruses and eukaryotic parasites, indicating that they can interact with other microorganisms sharing the same host environment. Using the terrestrial isopod crustacean Armadillidium vulgare, its highly diverse microbiota  and its three feminizing Wolbachia strains  as a model system, the present study demonstrates that Wolbachia can even influence the composition of a diverse bacterial community under both laboratory and natural conditions. While host origin is the major determinant of the taxonomic composition of the microbiota in A. vulgare, Wolbachia infection affected both the presence and, more importantly, the abundance of many bacterial taxa within each host population, possibly due to competitive interactions. Moreover, different Wolbachia strains had different impacts on microbiota composition. As such, infection with wVulC affected a higher number of taxa than infection with wVulM, possibly due to intrinsic differences in virulence and titer between these two strains. In conclusion, this study shows that heritable endosymbionts such as Wolbachia can act as biotic factors shaping the microbiota of arthropods, with as yet unknown consequences on host fitness. These effects can be beneficial, e.g. providing essential nutrients lacking from the host’s diet or defence against natural enemies11 or parasitic, including reproductive parasitism14.Heritable symbiotic bacteria are essential drivers of arthropod ecology and evolution. This is exemplified by the many species harbouring obligate or facultative vertically transmitted endosymbionts and the diversity of symbiont effects on host fitnessWolbachia are probably the most widespread heritable bacterial endosymbionts, infecting a wide range of arthropods (up to 65% of insect species are estimated to be infected16) and filarial nematodes17. Despite several cases of mutualism or dependence23, most arthropod-infecting Wolbachia are reproductive parasites: Being maternally transmitted, they manipulate their hosts’ reproduction in various ways (i.e. cytoplasmic incompatibility (CI), parthenogenesis, male-killing or the feminization of genetic males) to promote their own vertical transmission28. Although far from being the only bacteria inducing these phenotypes, Wolbachia are the most frequently encountered reproductive manipulators and cause the largest spectrum of reproductive phenotypes29.Bacteria of the genus 34. From this perspective, symbioses are shaped by highly dynamic multipartite interactions, not only between the host and its symbionts but also between the different members of the symbiotic community39. The latter may be direct interactions, e.g. through competition for resources or space within the shared host41 or by promoting the evolution of cooperation or dependence between different bacteria44. Alternatively, particular taxa could provoke a host immune response, which in turn might affect the microbiota as a whole, with potential consequences for organismal function. Indeed, studies of the relatively simple gut microbiota of Drosophila melanogaster have revealed (i) the importance of certain commensal bacteria for larval growth and optimal nutrient metabolism as well as (ii) a fine-tuned equilibrium between the host’s innate immune response and the gut microbiota to maintain homeostasis and prevent the proliferation of bacterial pathogens49. This leads us to ask whether heritable intracellular bacterial symbionts such as Wolbachia, which can reach high titers in host tissues and have profound impacts on host fitness, may also interact with the rest of the bacterial community present in the same host. This is all the more relevant since, due to its wide arthropod host range, Wolbachia is a dominant member of the microbiota in many insects, including species of agricultural or medical importance such as whiteflies or mosquitoes55.In addition to our growing understanding of the diversity of symbiont-mediated effects on hosts, the focus of symbiosis research has recently broadened from the study of binary host-symbiont interactions to a more holistic view of a host and its associated microbial communityWolbachia symbioses in arthropods, numerous studies have demonstrated a Wolbachia-mediated protection against various viruses and Plasmodium parasites in naturally infected Drosophila as well as in transfected mosquito species62. This protective phenomenon indicates that Wolbachia can indeed interact with other microorganisms in the same host environment, either indirectly via the induction of a general immune response, or via competition for resources and space. In contrast, relatively little is known regarding interactions between Wolbachia and other bacteria, except for several binary interactions with other highly abundant symbionts. Hence, male-killing Spiroplasma have been shown to negatively affect Wolbachia titers in D. melanogaster40 and recent studies have demonstrated a mutual competitive exclusion between Wolbachia and Asaia in the reproductive organs of Anopheles and Aedes mosquitoes, effectively inhibiting vertical transmission of the respective other symbiont64. However, Wolbachia’s influence on a larger commensal bacterial community has been investigated only recently in laboratory lines of D. melanogaster66, yielding conflicting results: While Wolbachia infection resulted in an overall decrease in taxonomic richness of the Drosophila gut microbiota, along with an increased abundance of the two bacterial families Leuconostocaceae  and Acetobacteraceae (Alphaproteobacteria) in Ye et al.66, Simhadri et al.65 instead observed significantly reduced titers of Acetobacteraceae, notably Acetobacter pasteurianus. These results indicate that Wolbachia-microbiota interactions may be complex and dependent on both host genotype and Wolbachia strain.In the context of Wolbachia on a diverse symbiotic bacterial community under both laboratory and natural conditions. To achieve this, we used the terrestrial isopod Armadillidium vulgare and its association with feminizing Wolbachia as a model system for several reasons: First, three different feminizing Wolbachia strains  establish stable single-infections in this host70, allowing us to compare the impact of different Wolbachia strains without having to account for additional interactions between the different Wolbachia strains themselves. Second, our recent quantitative study revealed Wolbachia strain-specific tissue distribution patterns in this species71, possibly reflecting different co-evolutionary histories between the Wolbachia strains and A. vulgare73. Third, the strain wVulC has recently been shown to protect its host against two bacterial pathogens74, akin to the protective phenotypes against viruses and parasites in Drosophila and mosquitoes, suggesting that the presence of this strain indeed affects co-infecting bacteria. Finally, a recent in-depth characterization of the microbiota in various tissues of A. vulgare unveiled a highly diverse bacterial community compared to many insect species, comprising more than 200 bacterial genera even in the presence of highly abundant Wolbachia75. Moreover, microbiota composition differed between host populations due to an important share of environmental bacteria, resulting in a complex community of intracellular and intestinal symbionts as well as environmental passengers75. Herein, we investigate the specific impact of Wolbachia infection on microbiota composition in A. vulgare using 16S rRNA gene metabarcoding and genetic fingerprinting via Temperature Gradient Gel Electrophoresis (TGGE). Our results show that, although host origin is the major determinant, Wolbachia infection has a noticeable and strain-specific impact on microbiota composition within each host population, resulting in reduced abundances of many bacterial taxa, possibly due to competitive interactions.The objective of the present study was to investigate the impact of A. vulgare collected from four laboratory lineages and two field sites in France , as well as wVulM-infected females (Availles only) and a single intersex male (the result of incomplete feminization) infected with wVulC from the Plaine Mothaise (Table Wolbachia strain) were pooled for sequencing. This resulted in 55 amplicon pools yielding 313 457 high-quality reads clustered into 1380 OTUs represented by ≥3 reads at the 97% similarity cut-off   Fig. . These rA. vulgare75, we first tested whether Wolbachia infection also had an impact on microbiota composition, independent of the host population . Principal Coordinates Analysis (PCoA) based on Bray-Curtis distances confirmed that host origin was indeed the major factor determining microbiota composition, with the first 2 principal components together explaining 63.71% of the variation  discriminated between Wolbachia-infected and uninfected isopods  had been reared under controlled environmental conditions, (ii) had received the same food sources, and (iii) harboured all three feminizing Wolbachia strains. Interestingly, 43.84% (267/609) of the OTUs observed in these specimens occurred exclusively in Wolbachia-infected females, while only 9.36% (57/609) of the OTUs were specific for uninfected males and females, respectively, and 18.23% (111/609) were shared between all samples, independent of gender and infection status  were observed specifically in females infected with wVulP, compared with females harbouring wVulC (N = 60) or wVulM (N = 67)  Fig. . These rWolbachia) were observed across all TGGE profiles, but none of these bands was found in all individuals. Indeed, the majority of bands occurred only in a relatively low number of individuals  and the band corresponding to Wolbachia was detected at the highest frequency, being present in the 20 infected females. This suggests a relatively high level of inter-individual variation in microbiota composition in A. vulgare. Between-Class-Analysis of these profiles based on gender and Wolbachia infection resulted in three distinct clusters corresponding to the microbiotas of uninfected males, uninfected females and Wolbachia-infected females , suggesting that both Wolbachia infection and host gender may influence microbiota composition. Unfortunately, since to date there is no reliable method to determine the genetic sex of A. vulgare, we could not determine which of the Wolbachia-infected individuals used in this study were indeed feminized genetic males.Considering that the sequencing data represented pooled amplicons from several individuals and therefore did not allow us to investigate inter-individual variations, we complemented the metabarcoding data with individual bacterial community profiles for the same specimens from the laboratory lineages using Temperature Gradient Gel Electrophoresis (TGGE) of the V3 region of the 16S rRNA gene. A total of 46 distinct bands , we identified bacterial genera which were either (i) present or absent depending on Wolbachia infection or (ii) present in both Wolbachia-infected and uninfected isopods but differentially abundant based on DESeq. 2 analysis76. This was done for each population separately, but also after combining the data from all host populations in order to identify more general patterns , Spirosoma (Bacteroidetes), Granulicatella (Firmicutes) and Cupriavidus (Betaproteobacteria)) and all four were only present in Wolbachia-free isopods. Despite the fact that the other genera did not occur in all host populations, several patterns emerged when looking at higher taxonomic levels: Hence, genera belonging to the phylum Cyanobacteria and to the classes Thermoleophilia (phylum Actinobacteria), Flavobacteria (Bacteroidetes) and Erysepilotrichi (Firmicutes) were only observed in Wolbachia-free isopods, while genera belonging to the phyla Gemmatimonadetes and Verrucomicrobia as well as to the classes Bacteroidia and Sphingobacteria (Bacteroidetes) were only observed in Wolbachia-infected specimens  were indeed systematically present or absent depending on ens Fig. . Moreoveods Fig. . Interesins Fig. . SimilarulP Fig. . In contion Fig. .Wolbachia infection across all populations, mostly belonging to the Actinobacteria (11 genera), Alphaproteobacteria (9 genera) and Gammaproteobacteria (8 genera) , Bacillus (Firmicutes), Ca. Hepatincola and Sphingomonas (Alphaproteobacteria), Ralstonia (Betaproteobacteria), Halomonas, Pseudomonas, Rickettsiella and Shewanella (Gammaproteobacteria) and Ca. Hepatoplasma (Tenericutes)). Both Wolbachia infection as well as different Wolbachia strains shaped bacterial abundance patterns, since more differentially abundant genera were detected in wVulC-infected isopods than in those harbouring wVulM . This was due to a higher number of differentially abundant genera belonging to the Actinobacteria and Gammaproteobacteria in wVulC-infected animals from the Plaine Mothaise and Availles, respectively  were found to be differentially abundant depending on ra) Fig. . In contely Fig. . Howeverely Fig. .Ca. Hepatoplasma crinochetorum (Mollicutes) and Ca. Hepatincola porcellionum (Alphaproteobacteria), both facultative extracellular symbionts of the midgut caeca79, and the bacterial pathogen Rickettsiella (Gammaproteobacteria)82. Each of these genera was identified as differentially abundant depending on Wolbachia infection in certain host populations: Ca. Hepatoplasma was found to decrease in abundance in Wolbachia-infected laboratory lineages (particularly in the lineages harbouring wVulM and wVulP), Ca. Hepatincola and Rickettsiella increased in abundance in wVulC-infected isopods from Availles and Rickettsiella decreased in abundance in Wolbachia-infected specimens from the Plaine Mothaise , while being almost absent from the two lineages derived from French populations75, which explains the observed statistically significant decrease in abundance. Moreover, a previous study found no differences in Hepatoplasma titer based on quantitative PCR between the Wolbachia-free and wVulC-infected laboratory lineages 75. Similarly, only a single male from the Plaine Mothaise population was infected with Rickettsiella while all females were found uninfected based on diagnostic PCR, which again explains the statistically significant decrease in abundance in Wolbachia-infected isopods from this population but not the increase in abundance in the Availles population, where Rickettsiella infection is more widespread in both males and females75. Hence, it remains to be investigated whether Hepatincola and Rickettsiella do indeed interact with Wolbachia.Several taxa need to be highlighted since they are well-known isopod symbionts: ise Fig. . HoweverArmadillidium vulgare harbours a highly diverse bacterial community consisting of ≥200 bacterial genera75 and is frequently infected by feminizing Wolbachia83. Apart from the reproductive manipulation, these Wolbachia have profound effects on host life history traits, e.g. reducing fecundity and immunocompetence73. In addition, the feminizing strain wVulC has recently been shown to protect its host against two bacterial pathogens74, indicating that Wolbachia can affect other co-infecting bacteria in this species. Using A. vulgare and its three feminizing Wolbachia strains  as a model system, the present study shows for the first time that Wolbachia not only interacts with certain bacterial pathogens, viruses or eukaryotic parasites, but can even influence a diverse bacterial community as a whole under both laboratory and natural conditions. While Wolbachia infection had no impact on bacterial taxonomic richness and diversity, it influenced the taxonomic composition of the microbiota and, more importantly, the abundance of many bacterial taxa. Thus, 48 genera , mainly belonging to the phyla Actinobacteria and Proteobacteria, were found to be differentially abundant depending on Wolbachia infection. Moreover, different Wolbachia strains also had different impacts on microbiota composition. In particular, a higher number of differentially abundant genera were detected in wVulC-infected isopods than in those harbouring wVulM, especially in specimens from a natural population in which both strains were present. This is of interest in light of previous studies suggesting that these two Wolbachia strains represent different co-evolutionary histories with A. vulgare and that wVulC has higher virulence, transmission rate and feminizing capacity than wVulM84. wVulM also reaches lower titers than wVulC in most host tissues71, which may explain both its lower virulence and the weaker impact on other bacterial taxa observed here. The wVulP strain is presumably the result of a recombination event between wVulC and wVulM, with wVulC as its major parent67. While wVulP has indeed similar tissue-specific titers as wVulC71, the microbiota of the laboratory-reared isopods harbouring this strain was clearly different from lineages infected with wVulC or wVulM, due to several genera belonging to the Actinobacteria and Alphaproteobacteria which were specifically associated with the wVulP lineage. Unfortunately, we did not find any isopods harbouring this strain in natural populations to corroborate this effect in a different host genetic background.The terrestrial isopod Wolbachia infection were low-abundance genera, making it less likely that the observed Wolbachia-mediated changes could be sufficiently strong to influence the performance of terrestrial isopods as keystone species in soil ecosystems. Interestingly, several better-studied and highly abundant terrestrial isopod facultative symbionts (Ca. Hepatoplasma crinochetorum (Mollicutes), Ca. Hepatincola porcellionum (Alphaproteobacteria) and Rickettsiella (Gammaproteobacteria)) were also identified as differentially abundant depending on Wolbachia infection. While this is likely a sampling artefact in the case of Ca. Hepatoplasma, potentially a nutritional symbiont enhancing host survival on low-quality diets85, it remains to be investigated using specific quantitative methods whether Ca. Hepatincola and Rickettsiella (a deadly isopod pathogen) are indeed more abundant in Wolbachia-infected individuals. If this were the case, it could suggest some kind of facilitation due to the presence of Wolbachia (e.g. reduction in host immunocompetence73) and the absence of Wolbachia-mediated resistance against the natural isopod pathogen Rickettsiella. Alternatively, Rickettsiella might reach higher titers in Wolbachia-infected individuals due to a Wolbachia-mediated tolerance as previously observed in D. simulans60, allowing the host to survive with higher pathogen loads compared to Wolbachia-free specimens.Nonetheless, it has to be pointed out that all the genera which were specifically present or absent and most genera identified as differentially abundant depending on Wolbachia-mediated protection against pathogens in Drosophila and mosquitoes, it has been hypothesized that these protective phenotypes might be due to (i) an enhanced immune response, especially in transfected non-native hosts87, or (ii) competition between Wolbachia and other microorganisms for resources and space in the shared host environment, resulting in titer-dependent protection90. Several factors make it more likely that the observed differences in the microbiotas of Wolbachia-infected A. vulgare are predominantly due to competitive interactions: (i) Previous studies demonstrated a reduction in several immune effectors in Wolbachia-infected isopods, arguing against immune priming73, (ii) total bacterial loads increase in some, but not all tissues of Wolbachia-infected individuals, suggesting a competition for resources or space between Wolbachia and other bacteria71, and (iii) in line with the latter, the present study shows that most differentially abundant bacterial phylotypes indeed decreased in abundance.Since the first discoveries of 75, Wolbachia infection is not the only factor shaping microbiota composition in A. vulgare. In particular, host origin had been previously identified as a major factor determining the taxonomic composition of the microbiota in this species75. While this could be due to both host genotype and/or different environments (e.g. in terms of soil or food-associated bacteria), we argue that the latter is the more important driver in A. vulgare. First, although the laboratory lineages used in this study were established from three different natural populations with very different genotypes71, their microbiotas are more homogenous in taxonomic composition after many years of controlled laboratory rearing on the same food sources than their conspecifics from natural populations75. This pattern was also obvious in the present study, since different bacterial presence/absence and differential abundance patterns were observed for each population. Second, environmental bacteria have an important share in the taxonomic richness of the microbiota in A. vulgare, potentially acquired from the prevailing food sources or the surrounding soil environment75. This has been corroborated by a recent study in the terrestrial isopod Porcellio scaber, whose microbiota composition was found to vary depending on different plant food sources in a controlled feeding experiment91. Host gender may be an additional confounding factor, especially in the present case where (i) only females harbour Wolbachia, (ii) it is impossible to distinguish Wolbachia-infected genetic females from feminized genetic males since no sex-specific markers are currently available for terrestrial isopods, and (iii) uninfected genetic females are rare in infected populations. Hence, it is impossible to determine which of the Wolbachia-infected specimens used in this study were genetically male or female, thus precluding any firm conclusions regarding the genetic sex as a driver of microbiota composition. Nonetheless, our data indicate that Wolbachia infection has a distinct impact on microbiota composition, independent of the genetic sex of the host: While the microbiota of uninfected females from the laboratory could not always be distinguished from that of uninfected males of the same genotype , the microbiota associated with wVulC-infected females from the same initial population was different from both uninfected males and females in both analyses. Moreover, the microbiotas of uninfected males from several populations were different from those of infected females , indicating a more general trend independent of host genotype, host genetic sex or environmental factors. Lastly, the microbiota of the Wolbachia-infected intersex male was most similar to the microbiota of infected females from the same natural population instead of clustering with the uninfected males.Based on the data presented here and in our previous studyA. vulgare appears to be shaped by several factors: Host origin , Wolbachia infection status and host gender. While the respective impacts of these factors are not easily disentangled, similarly complex multifactorial patterns likely underlie many animal-bacteria symbioses under ecologically realistic conditions.Taken together, the composition of the bacterial microbiota associated with Armadillidium vulgare used in this study were sampled from four laboratory lineages and two field sites in France . The Wolbachia-free lineage and the wVulC lineage were established from the same original population sampled in Helsingör, Denmark in 1982. The other two lineages carrying wVulM or wVulP derive from specimens sampled in Mery-sur-Cher (France) in 1999 and in Poitiers (France) in 2007, respectively. 10 males and 10 females (pairs of brothers and sisters) were randomly chosen from the Wolbachia-free lineage, as well as 10 females each from the wVulC, wVulM and wVulP lineages. Additional isopods from two natural populations in France  and Plaine Mothaise ) were sampled in autumn 2011 and 2012. The collected specimens were kept in plastic boxes with soil and leaves from their respective sampling site until dissection.The 77 ce Table  and were92. Wolbachia infection status as well as Wolbachia titers in all host tissues of the specimens used in this study have been determined previously using diagnostic as well as quantitative PCR of the wsp gene75. Females collected from Availles harboured either wVulC or wVulM, while all females collected from the Plaine Mothaise were infected with wVulC. In addition, one individual from the Plaine Mothaise was identified as an intersex male infected with wVulC: While the external morphological characters were male, the androgenic glands were hypertrophied, which is an indication of Wolbachia infection with incomplete feminization94.Prior to dissection, all isopods were surface-sterilized using sodium hypochlorite and haemolymph was collected after piercing the dorsal cuticle with a sterile needle. For each specimen, DNA was extracted from the collected haemolymph and four different tissues  using phenol–chloroformA. vulgare in different host tissues and populations75 (Accession Number PRJEB8160). Briefly, a 526 bp-fragment spanning the variable regions V1-V3 of the 16S rRNA gene was amplified using the universal primers 27 F and 520 R. Primers were adapted for 454 pyrosequencing by adding the 454 Adapter A and a 10-bp Multiplex Identifier sequence (MID) to the reverse primer 520 R as well as the 454 Adapter B to the forward primer 27 F. Amplicons were obtained from five different tissues  and up to 10 biological replicates from the same tissue and sample type (origin x gender x Wolbachia strain) were pooled for sequencing, resulting in 55 amplicon pools . Briefly, the flowgrams were denoised with AmpliconNoise97 and chimeras were removed using Perseus97. All reads shorter than 250 bp were discarded and the remaining reads were clustered into Operational Taxonomic Units (OTUs) at 97% similarity using uclust98. Representative sequences were aligned against the Silva reference alignment  using PyNAST100 and identified using the RDP Classifier101. Rare OTUs (i.e. singletons and doubletons) were discarded, resulting in 1380 OTUs represented by ≥3 reads  based on Bray-Curtis distances. Venn diagrams were produced in R using the VennDiagram package. Differentially abundant taxa were determined after data normalization using the DESeq. 2 Wald Test76 as implemented in QIIME.The 16S rRNA gene pyrotags were analysed using QIIME version 1.9.1102, followed by a second amplification targeting the V3 region using primers 338F-GC and 520 R103. Primer 338 F contained a 42-nucleotide GC-clamp preventing the complete denaturation of the DNA strands during TGGE103. All PCR amplifications were confirmed by electrophoresis in 1.5% agarose gels. TGGE was performed using the DCode Universal Mutation Detection System™ (Bio-Rad) following a protocol modified from104. Briefly, 20 μl of the final PCR products were run across a temperature gradient from 38 °C to 70 °C (ramping of 1.3 °C/h) at 60 V on 10% polyacrylamide gels. A standard containing V3 16S rRNA gene fragments from several reference bacteria  was loaded on each gel to allow the standardization of bands between gels. This also allowed a visual screening of Wolbachia infection across all samples via the presence of a band at the position corresponding to Wolbachia spp. in the standard. After electrophoresis, gels were stained with ethidium bromide and photographed under UV. Banding patterns on each gel were standardized based on the position of the reference bands using the GelAnalyzer 2010 software . Banding patterns across all gels were then analysed using an in-house Perl script: Bands with highly similar positions across all gels were grouped into a single normalized band, resulting in a presence/absence matrix of each normalized band per tissue sample. Finally, bacterial community profiles of each individual were established by merging the tissue-specific profiles into a single presence/absence profile per individual. The band corresponding to Wolbachia was removed from the dataset and bacterial community composition was analysed via principal component analysis (PCA) followed by between-class analysis (BCA)105 in R using the ade4 package. The 3D image of the BCA was made using the function scatter3d of the car package.Considering that the sequencing data from pooled amplicons did not allow us to investigate inter-individual variations, the metabarcoding data was complemented with individual bacterial community profiles from the same specimens from the laboratory lineages using Temperature Gradient Gel Electrophoresis (TGGE). A 196 bp-fragment of the variable region V3, also included in the fragment used for amplicon sequencing, was amplified from the tissue samples using a nested PCR approach: First, a 795-bp fragment was amplified using primers 27 F and 786 RThe 16S rRNA gene dataset used in this study is accessible in the European Nucleotide Archive under the Accession Number PRJEB8160.Table S1, S3 & S4Table S2Supplementary information Figure S1