An uncommon form of CNS tuberculosis called non‐osseous IDEM tuberculoma frequently results from paradoxical drug interactions. It should be considered one of the differentials when patients receiving ATT experience acute neurological impairment.Tuberculoma affecting the spinal cord is a rare condition in modern times. The occurrence of non‐osseous intradural tuberculosis, specifically in the spine, is even more exceptional. In fact, it is uncommon to encounter an intradural extramedullary tuberculous granuloma that lacks radiological indications of vertebral involvement, especially within the thoracic region. We present a case of a patient with a neurological deficit caused by a non‐osseous intradural tuberculoma in the thoracic region, without any associated bone involvement. The patient experienced a gradual deterioration of neurological function. An MRI of the thoracic spine revealed the presence of a tuberculoma located intradurally, extramedullary, and juxtamedullary of the T5 vertebra. The compression of the spinal cord resulted in paraparesis which was worsening to paraplegia. A D4–D6 laminectomy and microsurgical excision were performed under intraoperative neurophysiological monitoring (IONM), and the patient showed clinical recovery. Excellent clinical outcomes were achieved. However, it is crucial to consider the possibility of a non‐osseous intradural tuberculoma as a rare condition when encountering a SOL, particularly in patients with a history of tuberculosis and spinal cord compression. In cases where a progressing neurological deficit is present, a combination of surgical intervention and anti‐tuberculous treatment should be considered as the optimal approach. The MRI images of the thoracic spine. (A) Coronal T1‐weighted MRI image shows a ring‐like enhancing intradural lesion after gadolinium. (B) Sagittal T1‐weighted MRI image shows a 2.1 × 1.7 × 1.45 cm intradural/juxtamedullary and extramedullary mass with central necrosis and marginal enhancement after gadolinium suggestive of immature tuberculoma at T5 level. (C) Axial T2‐weighted image. The CNS involvement in developing countries constitutes nearly 10% of all tuberculosis patients.Here we will be discussing a case of intradural extramedullary tuberculosis of the thoracic spine, with progressive neurological dysfunction. Surgery aims to decompress the spinal cord and remove the tuberculoma, thereby relieving the pressure on the neural tissues and preventing further neurological deterioration.Following surgery, anti‐tubercular therapy is initiated to target the underlying tuberculosis infection. This typically involves a combination of multiple anti‐tuberculosis medications, such as isoniazid, rifampicin, ethambutol, and pyrazinamide.This report aims to discuss the diagnostic challenges, treatment strategy, and clinical outcomes, highlighting the importance of a multidisciplinary approach in managing this uncommon condition.2A 29‐year‐old man, who had been under treatment for disseminated tuberculosis for 7 months, was admitted with progressive weakness of both lower limbs leading to difficulty in walking. There was no history of fever, cough, palpitations, or breathlessness. Physical examination revealed a palpable spleen but no lymphadenopathy. He had spastic paraparesis with involvement in right side more than the left. There was severe girdle pain at T5 level. The patient underwent a computed tomography (CT) chest, which revealed multiple patchy resolving ground glass opacities and interlobular septal thickening in bilateral lower lobes, predominantly superior and posterior basal segments, suggesting resolving tuberculosis. Also, mild centrilobular emphysema in bilateral upper lobes with sub‐centimeter right paratracheal and left paraaortic lymph nodes, the largest measuring 7 mm, were present. Other findings included splenomegaly measuring 17.6 cm and calcified hepatic granulomas (segment VII).Magnetic Resonance Imaging (MRI) thoracic spine revealed a single 2.1 × 1.7 × 1.45 cm intradural/juxtamedullary and extramedullary mass appearing as well‐defined T1‐weighted low signal/T2‐weighted FLAIR bright signal with central necrosis and marginal enhancement after gadolinium (Gd) suggestive of immature tuberculoma at T5 level with surrounding edema Figure .No evidence of vertebral TB, spinal tract infiltration, or engulfment was noted. A diagnosis of D5 intradural extramedullary space‐occupying lesion (SOL) with spastic paresis, cord compression, compressive myelopathy, disseminated tuberculosis, and post‐tuberculous medication‐induced hepatic granuloma was made. D4‐D6 laminectomy and microsurgical excision under IONM were planned to remove the lesion. After pre‐anesthetic assessment and consent, the patient underwent the procedure with the head held in a horseshoe, subperiosteal muscle dissection made, and laminae exposed. Durotomy was performed under a microscope, and the lesion was visualized. A grayish‐white, firm lesion with a central necrotic area was exposed Figure .The pus was aspirated, followed by internal bulking using ultrasonic surgical aspirator Figures , 4, 5. T3Tuberculosis (TB) remains a common health issue in developing countries, even though TB has not been seen widely as before.M. tuberculosis neuropathogenesis. Although it helps in granuloma formation and control of mycobacterial infections, local production of TNF‐α in the central nervous system can alter the permeability of the blood–brain barrier and promote the progression of TBM.The pathogenesis of central nervous system tuberculosis involves the development of small foci of tuberculosis, known as rich foci, in the brain, spinal cord, or meninges.M. tuberculosis, and the CD14 receptor facilitates engulfment of unopsonized bacilli by microglia. Microglia infected with M. tuberculosis produce a variety of cytokines and chemokines, including TNF‐α, IL‐6, IL‐1β, CCL2, CCL5, and CXCL10. Microglia play a central role in the neuropathogenesis of tuberculosis, and their infection can lead to immunosuppressive effects, especially in more virulent strains.Human microglia are selectively infected by Similarly, among various forms of spinal TB involvement, such as Pott disease, non‐osseous spinal TB, tuberculous spinal meningitis, and tuberculous arachnoiditis, non‐osseous spinal tuberculoma is infrequent. In a review conducted by Dastur, 74 cases of spinal tuberculomas were analyzed, revealing that 65% were located extradurally, 8% were intramedullary, 5% were intradural extramedullary, and 20% were arachnoidal.The clinical presentation of the case aligns with the findings reported in the literature, which often show a higher prevalence among males.A combination of surgical intervention and medical treatment has shown excellent outcomes. While some authors argue that anti‐tuberculous therapy alone is sufficient when a paradoxical reaction develops into a tuberculoma,The list of similar published cases is mentioned in Table 4IDEM tuberculoma is a rare manifestation of CNS tuberculosis, often arising as a result of paradoxical reactions to anti‐tubercular medication. When patients on anti‐tubercular therapy develop new‐onset neurological deficits indicative of compressive myelopathy, IDEM tuberculoma should be considered as one of the potential causes. Early and accurate diagnosis, along with timely initiation of appropriate medical or surgical interventions, is crucial to prevent irreversible dysfunction. Excision plays a pivotal role in the treatment of this uncommon condition.Shyam Duvuru: Conceptualization; data curation; formal analysis; investigation; methodology; writing – original draft; writing – review and editing. Vivek Sanker: Formal analysis; investigation; methodology; project administration; resources; supervision; validation; visualization; writing – original draft; writing – review and editing. Syed Naureen: Resources; software; supervision; validation; visualization; writing – original draft; writing – review and editing. Gupta Prakash: Software; supervision; validation; visualization; writing – original draft; writing – review and editing. Rajurkar Sanjana: Resources; software; supervision; validation; visualization; writing – original draft; writing – review and editing. Tirth Dave: Resources; software; supervision; validation; visualization; writing – original draft; writing – review and editing.None.None declared.The ethical approval was not required for the case report as per the country's guidelines.Written informed consent was obtained from the patient to publish this report.


Aggressive tumor‐signature mtDNA mutation detection and measurement of mtDNA and CL contents in the EVs bear the potential to formulate noninvasive early detection and recurrence prediction strategies.Early detection and recurrence prediction are challenging in triple‐negative breast cancer (TNBC) patients. We aimed to develop mitochondrial DNA (mtDNA)‐based liquid biomarkers to improve TNBC management. Mitochondrial genome (MG) enrichment and next‐generation sequencing mapped the entire MG in 73 samples (64 tissues and 9 extracellular vesicles [EV] samples) from 32 metastatic TNBCs. We measured mtDNA and cardiolipin (CL) contents, NDUFB8, and SDHB protein expression in tumors and in corresponding circulating EVs. We identified 168 nonsynonymous mtDNA mutations, with 73% (123/186) coding and 27% (45/168) noncoding in nature. Twenty percent of mutations were nucleotide transversions. Respiratory complex I (RCI) was the key target, which harbored 44% (74/168) of the overall mtDNA mutations. A panel of 11 hotspot mtDNA mutations was identified among 19%–38% TNBCs, which were detectable in the serum‐derived EVs with 82% specificity. Overall, 38% of the metastatic tumor‐signature mtDNA mutations were traceable in the EVs. An appreciable number of mtDNA mutations were homoplasmic , novel , and potentially pathogenic . The overall and RCI‐specific mtDNA mutational load was higher in women with African compared to European ancestry accompanied by an exclusive abundance of respiratory complex (RC) protein NDUFB8 (RCI) and SDHB (RCII) therein. Increased mtDNA  contents. Circulating mtDNA mutation detection and measurement of mtDNA and CL contents bear the potential to formulate noninvasive early diagnostic and recurrence prediction strategies. In addition, circulating sera‐derived EVs from nine of these patients were also sequenced for mtDNA mutation detection, therefore, a total of 73 samples were analyzed. We have detected 168 nonsynonymous mtDNA mutations in these patients spanning the coding, and noncoding regulatory regions of the MG. Interestingly, mtDNA mutations were detectable in TNBC patients with lymph node (LN) metastasis. Respiratory complex I (RCI) appears to be the mutational hotspot in these patients. The key and hotspot mtDNA mutations were readily detectable in the circulating EVs. We detected an additional 11 mtDNA mutations, exclusively in the EVs of the nine TNBC patients we examined. We also identified a considerable number of homoplasmic, novel, and potentially pathogenic mtDNA mutations in these patients. The overall and RCI‐specific mtDNA mutational burden was higher in the African compared to the European‐TNBC patients, who also exhibited exclusive overexpression of nDNA encoded RCI‐NDUFB8 RCII‐SDHB proteins. In addition, we detected enhanced mtDNA contents in the TNBC tumors and the EVs compared to their normal counterparts. An abundance of cardiolipin (CL) content was also recorded in the circulating EVs of the TNBC patients compared to the cancer‐free control subjects.22.1We obtained de‐identified archived tumors and matched normal tissue samples from 30 triple‐negative breast cancer patients from the Biospecimen Repository Core Facility (BRCF) at the University of Kansas Medical Center (KUMC). Of the 30 cases, 21 cases were formalin‐fixed paraffin‐embedded (FFPE) tissue samples and nine were fresh‐frozen tissue samples. Paired normal/tumor samples (frozen) from additional two TNBC patients were also collected from the Cooperative Human Tissue Network (CHTN). Matched sera samples from nine patients from the BRCF were also collected. Additional serum samples from nine cancer‐free individuals were also collected. The studies using human specimens were approved by the IRB (protocol #20‐222) at the University of South Alabama. Samples were stored at −80°C or 4°C as appropriate until further use. All the de‐identified subjects have consented to allow for the use of their specimens, and only relevant clinical information including age, race, primary diagnosis, stage, grade, etc., was collected for statistical analyses. All methods were performed following the relevant guidelines and the regulations of the University of South Alabama. The demographic information of all the subjects along with the type of samples utilized from each patient is presented in Table 2.2n = 22, Table n = 42), we used seven 10‐μm curls followed by gDNA isolation using the QIAamp DNA FFPE Tissue Kit and protocol . DNA isolation was carried out on the automated nucleic acid extraction station QIAcube Connect (Qiagen) and DNA integrity was checked using QIAxcel DNA Screening Kit on the QIAxcel advanced instrument. In the next step, mtDNA was enriched from all the samples (n = 64) using the REPLI‐g Mitochondrial DNA Kit and protocol  as described.For genomic DNA (gDNA) isolation from the frozen normal/tumor samples . From the gDNA pool of each patient, we enriched mtDNA using REPLI‐g Mitochondrial DNA Kit (QIAGEN), which utilizes multiple displacement amplification technologies.2.5n = 73). Variant Pro Amplicon Sequencing (Variant Pro™) platform was used for NGS of the entire MG (Table https://www.ncbi.nlm.nih.gov/nuccore/251831106) using Bwa‐0.7.12. The alignment was corrected using GATK‐3.8‐0, the variant calling was conducted using ATK‐3.8‐0, Samtools‐1.9, and Varscan‐v2.4.3, and the annotation of the variants was performed using Annovar‐201,707 and snpEff‐4.3i. For a variant call, all the resulting mtDNA sequence variants were interrogated at different human MG databases as described.We utilized 200 ng of highly enriched mtDNA for subsequent next‐generation sequencing (NGS) analysis from all the samples  membrane (Sigma Aldrich) and subjected to standard immunodetection procedure using specific antibodies: NDUFB8 (1:1000) , SDHB (1:1000) , and β‐actin (1:20000) . Secondary antibodies  were used at 1:2000 dilutions. Blots were processed with SuperSignal West Femto Maximum Sensitivity Substrate Kit  and the signal was detected using a ChemiDoc Imaging System (Bio‐Rad). Densitometry was done using Fiji (ImageJ) software from the National Institutes of Health.Protein extraction and western blotting were performed following a method as described earlier.2.7NDUFB8 and SDHB in TNBC and various other breast cancer subtypes using publicly available datasets from The Cancer Genome Atlas (TCGA) using a web‐based application UALCAN, the University of Alabama .We examined the mRNA expression pattern of 2.8n = 64) by real‐time quantitative PCR. To amplify mtDNA, we used MG‐encoded ND1 (MT‐ND1), MT‐CYTB, and nDNA‐encoded GAPDH, as described earlier.MT‐ND1/GAPDH and MT‐CYTB/GAPDH) ratios from triplicate wells of each subject were utilized to plot relative abundance in different groups. In addition, we also amplified mtDNA in the sera‐EVs from nine TNBC and nine cancer‐free control subjects using the multiple displacement amplification as described above using equal amounts of gDNA template. MtDNA fold enrichment was determined by the Qubit fluorometer quantitation system (ThermoFisher).We measured mtDNA content using gDNA (20 ng) from matched normal and tumor tissues of the 32 TNBC patients  in the EVs isolated from the sera of nine TNBC patients and nine cancer‐free subjects as described earlier.2.10t‐tests χ2, one‐way ANOVA or Fisher's exact tests as appropriate for statistical analyses. The p‐values were two‐sided with confidence intervals at the level of 95%. Computation for all the analysis was performed using the GraphPad Prism program.We employed Student's 33.1p = 0.62 and 0.91, respectively).We sequenced mtDNA in 73 tissue samples collected from 32 women with a primary diagnosis of TNBC. The quality of the sequence reads and the tumor MAFs of all the samples have been presented in Figure 3.2RNR1 and RNR2 and ~ 12% (20/168) mutations spanning the tRNA genes. The frequency of the RNR1 and RNR2‐mtDNA mutations in the TNBC patients was 3% except for one mutation (G1888A), which was detected in 6% of patients , RCIII (MT‐CYTB), RCIV , and RCV (MT‐ATP6 and ATP8) subunits. Among these coding mtDNA mutations, 44% (74/168) were from the RCI  were detected in the RCIII region represented by a single gene MT‐CYTB  mtDNA mutations in the noncoding regulatory region involving s Figure . In comps Figure . The resI Figure . Among tB Figure . The fren Figure . The fren Figure . Of the 3.3As mentioned above, 168 different types of somatic mtDNA mutations were detected in 32 unique TNBC cases. In the next step, we determined the frequency of occurrence of these mutations across different patients. We identified a panel of 11 hotspot mtDNA mutations, which were detected in 19%–38% of the patients Figure . Among tMT‐ND1) appeared to be novel.To identify whether tumor‐signature mtDNA mutations are traceable in the circulation, we enriched and sequenced mtDNA from sera‐derived EVs from nine of the TNBC cases Table . These n3.4NDUFB8 expression had African ancestry and were from Stages I‐III  in breast cancer patients (n = 1097) compared to their normal counterparts (n = 114)  was also noted in TNBC patients (n = 116) compared to the same normal control samples  in the African (n = 179) compared to the Caucasian (n = 748) breast cancer patients  was higher compared to the normal (n = 114), however, it did not reach a significant level . In comparison, a similar pattern of SDHB mRNA expression was evident in the breast cancer patients with African (n = 179) and European (n = 748) ancestry , which is nuclear‐encoded and involved in mitochondrial RCI assembly. In parallel, we also measured the expression pattern of SDHB (Succinate Dehydrogenase Complex Iron Sulfur Subunit B), a key subunit of RCII. Of the eight TNBC patients that we have examined, markedly higher expression of NDUFB8 was noted (3‐ to 56‐folds) in 50% (4/8) of the patients Figure . NotablyII Table  similar II Table . InteresII Table . NDUFB8 II Table , was appII Table . At the ) Figure . A signis Figure . Notablys Figure . On the l Figure . Howevery Figure .3.5MT‐ND5 and MT‐ND1 genes. Mutation load in MT‐ND1 and MT‐ND5 was higher in the African compared to the European‐TNBC patients   were shared between the TNBC patients with African and European ancestry. Similar was the case for the T4434G mutation in the tRNA‐Met gene.Among the 32 TNBC subjects, seven were of African ancestry, whereas 25 patients had European ancestry. Considering the overall mtDNA mutations, the mutational load was higher among the African‐TNBC compared to the European‐TNBC patients (7.5 vs. 4.6 mutations) Figure . We have) Figure . Notably3.6p < 0.0001) in the tumors of the TNBC subjects compared to their adjacent normal tissues   in the EVs of the nine TNBC patients compared to the cancer‐free subjects  Figure . We alsos Figure .3.7G8519A‐MT‐ATP8, C6340T‐CO1, C7758T‐MT‐CO2, and G8078A‐MT‐CO2) appeared to be potentially pathogenic in nature  were homoplasmic in nature. Among the noncoding mutations, 13% each (4/31) were detected in both the control Figure  and tRNAe Figure . The ove4EGFR, KRAS, P53, etc. are well established in human tumorigenesis.Despite recent advancements in the “multi‐omics” understanding of tumorigenesis, early detection of the lethal lesions, their recurrence prediction, and therapeutic management of TNBC patients are significant clinical challenges. The majority of TNBC patients are diagnosed at advanced stages, predominantly in young women with African ancestry.TNBCs are diagnosed predominantly at times when tumors already metastasized to the LN(s) and/or various organ sites. Even, the detection of a Stage‐I TNBC tumor with a minimum single LN metastasis is challenging due to the lack of appropriate interventions. Moreover, a Stage‐I tumor does not rule out the possibility of micro‐metastases in multiple LNs and distant organs, which remains undetectable by mammography and currently available methods at the time of first diagnosis. In addition, there are no reliable prognostic biomarkers for accurate monitoring of TNBCs. In this light, frequent detection of potentially pathogenic and hotspot mtDNA mutations in the tumors and the circulating EVs of the TNBC patients with the early onset of metastasis may serve as useful biomarkers for early detection of the lethal lesions, their monitoring, and measuring therapeutic response. At the same time, the detection of the frequently occurring mtDNA mutations in early metastatic TNBCs suggests their potential role in the recurrent and metastatic progression of the disease. Since TNBCs do not progress through the preneoplastic ductal carcinoma in situ (DCIS) in most of the cases and remain undetected without metastasis, we could not analyze those samples for mtDNA mutation detection. However, if the hotspot mtDNA mutations that we cataloged are associated with metastatic dissemination at the earliest time point, they can guide in predicting disease outcomes in advance, leading to improved therapeutic management.MT‐ND1 that we detected in the metastatic‐TNBC patients, was found to be associated with hepatocellular carcinoma progression.NDUFB8, exclusively in the primary tumors and established cell model derived from TNBC patients with African ancestry may have functional implications in racially disparate disease outcomes. Further comprehensive analysis of the variants is thus warranted. Evident loss of RCII‐SDHB expression in one patient reflects heterogeneity between tumor cell clones, which necessitates the development of personalized treatment. Although the noncoding genes do not make proteins, however, mutations in these genes can influence the expression of the coding genes and mitochondria‐nuclear retrograde signaling.In the OXPHOS system, RCI serves as the entry gate for the electron transport system to begin.EGFR gene mutation is present in 10%–20% of the patients with African and European ancestry and thereby serves as an excellent biomarker and therapeutic target.Recent studies suggest that transversion mutations have more potential to hamper transcription factors' binding, thereby causing profound changes in genes' expression.MT‐ND1), A10935G (MT‐ND4), T12922G, T13762G, A13780G (MT‐ND5), and A1811G (RNR2) were readily detectable in the tumors and matched EV samples of the metastatic‐TNBC patients. Thus, routine analysis of mtDNA mutation detection in EVs from cancer patients of various anatomic origins could be useful for accurate monitoring/surveillance and measuring therapeutic responses. Another study demonstrated the transfer of wild‐type MG through EVs from cancer‐associated fibroblasts to the hormone treatment‐resistant, and dormant breast cancer cells, thereby facilitating their progression.Early detection, accurate monitoring, and/or surveillance of cancer patients using a noninvasive or minimally invasive approach without requiring the tumor tissues at different time points are ideal for suitable disease management in clinical settings. In this context, circulating EV harboring metastatic tumor‐associated hotspot mtDNA mutations with high specificity could be excellent resources for minimally invasive liquid biomarker development. In a recent study, we detected several tumor‐associated coding and noncoding mtDNA mutations in sera‐derived EVs from pancreatic cancer patients.An increase in mtDNA content was shown to promote colorectal cancer progression by augmenting OXPHOS function.In summary, this is the first study cataloging the outcome of entire MG sequencing in tumor tissues and circulating EVs from metastatic‐TNBC patients with divergent ancestral backgrounds. Comprehensive profiling of the panel of hotspot mtDNA mutations in the tumors and their highly sensitive and accurate detection in the circulation of a larger TNBC cohort are warranted. Alongside periodic mammographic evaluations, the formulation of a circulating mtDNA mutation detection system in concert with the measurement of mtDNA and CL contents from a small amount of samples is feasible. This cost‐effective simple approach could open novel avenues for the early detection of potentially lethal lesions, their recurrence prediction, and the prevention of metastatic progression particularly in racially disparate populations.K.S. Vikramdeo: Data curation, investigation, methodology manuscript editing. S. Anand: Data curation, methodology, and manuscript editing. S.K. Sudan: Data curation, methodology, and manuscript editing. P. Pramanik: Data curation, methodology, reviewing, and manuscript editing. S. Singh: manuscript editing, reviewing, and investigation. A. Singh: Manuscript editing, reviewing, and investigation. A.K. Godwin: Manuscript editing, reviewing, resources, and investigation. S. Dasgupta: Conceptualization, data curation, formal analysis, resources, supervision, funding acquisition, investigation, methodology, writing original draft, reviewing and editing, and project management.This research was supported by funding from the Breast Cancer Research Foundation of Alabama (BCRFA), Mitchell Cancer Institute and the University of South Alabama to SD. The BRCF is supported in part by the KU Cancer Center's Support Grant (P30 CA168524). The authors would also like to thank all the patients for providing their precious samples.The authors declare no conflicts of interests.The studies have been approved by the appropriate institutional research ethics committee and have been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The BRCF was established to obtain human biospecimens from patients undergoing treatment at KUMC as part of a protocol approved by their internal Human Subjects Committee (HSC #5929).Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Table S1.Click here for additional data file.Table S2.Click here for additional data file.


Prophantis species in the tribe Trichaeini (Lepidoptera: Crambidae) were sequenced using high‐throughput sequencing technology. They were assembled and annotated: The complete mitogenomes of P. octoguttalis and P. adusta were 15,197 and 15,714 bp, respectively, and contain 13 protein‐coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and an A + T‐rich region. Their arrangement was consistent with the first sequenced mitogenome of Bombyx mori (Bombycidae) in Lepidoptera, which had the trnM–trnI–trnQ rearrangement. The nucleotide composition was obviously AT‐biased, and all PCGs, except for the cox1 gene (CGA), used ATN as the start codon. Except for trnS1, which lacked the DHU stem, all tRNA genes could fold into the clover‐leaf structure. The features of these two mitogenomes were highly consistent with those of other species of Spilomelinae in previous studies. Phylogenetic trees of Crambidae were reconstructed based on mitogenomic data using maximum likelihood and Bayesian inference analysis methods. Results showed that Trichaeini in this study robustly constitute a monophyletic group in Spilomelinae, with the relationships (Trichaeini + Nomophilini) + ((Spilomelini + (Hymeniini + Agroterini)) + Margaroniini). However, the affinities of the six subfamilies Acentropinae, Crambinae, Glaphyriinae, Odontiinae, Schoenobiinae, and Scopariinae within the “non‐PS Clade” in Crambidae remained doubtful with unstable topologies or low supports.The complete mitochondrial genomes of two  Prophantis species in the tribe Trichaeini were provided for the first time. Phylogenetic trees of Crambidae were reconstructed based on mitogenomic data using maximum likelihood and Bayesian inference analysis methods. Results showed that Trichaeini in this study robustly constitute a monophyletic group in Spilomelinae, with the relationships (Trichaeini + Nomophilini) + ((Spilomelini + (Hymeniini + Agroterini)) + Margaroniini).The complete mitochondrial genomes of two  P. octoguttalis, the type species of the genus, is widespread, and is mainly distributed in southern China, Australia, India, and the Afrotropical region , and a single larva can harm several berries in succession, which can seriously impact coffee production  is a closed‐loop DNA double‐helix molecule that varies significantly in length among taxa. The mtDNA of lepidopteran insects is generally 15–16 kb in size and consists of 37 genes, including 13 protein‐coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a control region of variable length also known as A + T‐rich region and D‐loop region Boore, . BecauseP. octoguttalis and P. adusta of the Trichaeini for the first time, and performed preliminary bioinformatics analysis, including the gene size and arrangement, base composition, codon usage, and tRNA secondary structure, which can help us to understand the features of mitogenomes of Trichaeini and Spilomelinae. To understand the phylogenetic relationships of Spilomelinae and the position of Trichaeini, we reconstructed the phylogenetic tree based on the mitogenomes data of these two species with other available mitogenomes of Crambidae from GenBank by using maximum likelihood (ML) and Bayesian inference (BI) methods. It will provide new perspectives and genomic data for the phylogenetic research in Trichaeini and Spilomelinae.To date, only 23 mitogenomes of Spilomelinae have been published in GenBank, and no mitogenomes of Trichaeini have been reported. In this study, we sequenced the mitogenomes of 22.1P. octoguttalis investigated was collected from Wuzhi Mountain in Hainan Province, China, in March 2021; the specimen of P. adusta was collected from Fanjing Mountain in Guizhou Province, China, in September 2020. Fresh specimens obtained by light trapping were soaked in anhydrous alcohol and stored at −80°C in the Insect Collection of Southwest University, Chongqing, China. DNA was extracted from the thoracic muscle of each specimen. The mitogenome was sequenced by BGI Genomics on Illumina next‐generation sequencing platform with Read Length of PE150 and Coverage depth of 6 G.The specimen of 2.2The high‐quality data (clean data) of the samples, which were trimmed by BGI Genomics, were saved as fastq. format and imported into analyzed on Geneious Prime v2022.1.1. The published COI sequences of each species (MH418217 and KY370920) were downloaded from GenBank as reference sequences . Sequence length, base composition, gene spacing, and overlap were viewed directly using Geneious Prime v2022.1.1. The base skew was calculated using the formula: AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C)  alignment was used to align the reference sequence with the sample sequence, and PCGs were determined based on the similarity between genes. With the help of EditSeq v7.1.0, PCGs were translated into amino acids to further verify the correctness of the start codon, stop codon, and amino acid sequence, to ensure the accuracy of PCGs. The location and secondary structure of tRNA genes were predicted using the MITOS Web Server  of Pyralidae, Bombyx mori of Bombycidae, and Helicoverpa armigera of Noctuidae were selected as outgroups  were used to construct the phylogenetic tree. The ingroups included five species of Acentropinae, five species of Crambinae, one species of Glaphyriinae, three species of Odontiinae, eight species of Pyraustinae, one species of Schoenobiinae, one species of Scopariinae, and 25 species of Spilomelinae. The four species (ps Table .We used two datasets: (1) PCG123: all three codon positions of 13 PCGs; (2) PCG123RT: all three codon positions of 13 PCGs, two rRNA genes and 22 tRNA genes. ML and BI were used to construct phylogenetic trees.7 generations, with simultaneous sampling every 1000 generations. The initial 25% of the sampled trees were discarded as burn‐ins. Chain convergence was assumed when the mean standard deviation of the split frequencies fell below 0.01. Bayesian posterior probability, in which the support of each node of the BI tree was greater than or equal to 0.95, was considered high confidence. The phylogenetic tree was constructed using Figtree v.1.4.4.ModelFinder , two rRNA genes (rrnL and rrnS), and eight tRNA genes  are encoded from the minority strands. The remaining 23 genes are encoded from the majority of the strands   is higher than that of the first (73.1% and 82.7%) and second codons (74.9% and 79.8%). The AT skew of these two species is 0.01 and 0.003, and their GC skew is −0.173 and −0.181, respectively, showing a slight A skew and an obvious C skew.Thirteen PCGs are identified in the mitogenomes of P. octoguttalis and P. adusta are 11,196 and 11,219 bp, encoding 3721 and 3728 amino acids, respectively. Statistics on the RSCU of P. octoguttalis and P. adusta show that the codons UUA(L), AUU(I), UUU(F), AUA(M), and AAU(N) are used most frequently. In P. octoguttalis, CUG, GUC, CCG, CGG, AGC, and AGG do not participate in amino acid synthesis, while in P. adusta, CUG and AGG do not participate. The codons of amino acids with RSCU >1 all contain A or U  are encoded by the majority chain, and the remaining eight genes are encoded by the minority chain, with the length of each gene ranging from 64 bp  to 71 bp. Except for trnS1 (AGN), which lacks the DHU stem, the secondary structures of the remaining 21 tRNAs fold into a typical clover‐leaf structure  gene occurs in the TψC stem.In the mitogenomes of these two species, there are 22 tRNA genes with concatenated lengths of 1468 and 1481 bp, respectively. A total of 14 genes , and the relationships within Spilomelinae were (Trichaeini + Nomophilini) + ((Spilomelini + (Hymeniini + Agroterini)) + Margaroniini). With the exception of the newly sequenced species of Trichaeini, the phylogenetic relationship among the tribes was roughly consistent with Liu et al.  with AgrThe differences among the four phylogenetic trees constructed in this study were mainly concentrated in the “non‐PS clade.” The “non‐PS clade” was divided into the “OG clade” and the “CAMMSS clade” (PP = 1/BS = 98) in the BI tree of dataset PCG123RT and the ML tree of dataset PCG123. The “OG clade” consisted of Glaphyriinae and Odontiinae, which were related to each other as sister groups, with a high to low support (PP = 1/BS = 55) and the monophyly of Odontiinae was highly supported (PP = 1/BS = 100). Acentropinae, Crambinae, Schoenobiinae, and Scopariinae formed the “CAMMSS clade,” which presented two close relationships, Acentropinae and Schoenobiinae as sister group (PP = 1/BS = 81), Scopariinae and Crambinae as sister group with a high to low support (PP = 0.93/BS = 57). This was consistent with the results in previous studies based on nuclear and mitochondrial markers  + ((Spilomelini + (Hymeniini + Agroterini)) + Margaroniini). Within the “non‐PS clade,” the monophyly of Acentropinae, Crambinae, and Odontiinae was well supported. The close relationship between Odontiinae and Glaphyriinae, between Schoenobiinae and Acentropinae, and between Scopariinae and Crambinae seemed to be more realistic.4Prophantis species, P. octoguttalis and P. adusta, belonging to the tribe Trichaeini, for the first time, and analyzed their gene size and arrangement, base composition, codon usage, and tRNA secondary structure, etc., which were highly consistent with those of other previously studied species of Spilomelinae. The two mitogenomes were typical of Lepidopteran insects. Combined with the published mitogenome sequences of Crambidae, all phylogenetic trees based on the different datasets confirmed the monophyly and position of Trichaeini and showed satisfactorily high support values. However, its sister group was not completely resolved, combined with previous multisite studies. In addition, the phylogenetic relationships within Crambidae in phylogenetic tree in our present study were in general agreement with previous studies, whereas the affinities in the “non‐PS clade” were still unstable and require further investigation. Therefore, improving sample coverage and combining different molecular markers, such as mitochondrial genome and nuclear genes, should be considered in the future research on these taxa.In this study, we reported the complete mitogenomes of two Ci Tang: Conceptualization ; data curation ; formal analysis (lead); methodology (lead); software (lead); writing – original draft (lead); writing – review and editing . Xicui Du: Conceptualization ; data curation ; funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); writing – review and editing .This work was sponsored by the National Natural Science Foundation of China (31772500) and by the Natural Science Foundation of Chongqing, China (No. CSTB2022NSCQ‐MSX1164).All authors declare no conflicts of interest.


In a series of studies using both archival and original data , we show that people in at least 60 nations around the world believe that morality is declining, that they have believed this for at least 70 years and that they attribute this decline both to the decreasing morality of individuals as they age and to the decreasing morality of successive generations. Next, we show that people’s reports of the morality of their contemporaries have not declined over time, suggesting that the perception of moral decline is an illusion. Finally, we show how a simple mechanism based on two well-established psychological phenomena (biased exposure to information and biased memory for information) can produce an illusion of moral decline, and we report studies that confirm two of its predictions about the circumstances under which the perception of moral decline is attenuated, eliminated or reversed . Together, our studies show that the perception of moral decline is pervasive, perdurable, unfounded and easily produced. This illusion has implications for research on the misallocation of scarce resources3, the underuse of social support4 and social influence5.Anecdotal evidence indicates that people believe that morality is declining We show that the perception of moral decline is pervasive, perdurable, unfounded and easily produced, and suggest that this illusion has implications for research on the misallocation of scarce resources, the underuse of social support and social influence. But as apt as this description of our times may seem, it was written more than 2,000 years ago by the historian Livy, who was bemoaning the declining morality of his fellow Roman citizens. From ancient to modern times, social observers have often lamented the ugly turns their societies have taken, and have often suggested that a recent decline in morality—in kindness, honesty and basic human decency—was among the causes7.The social fabric appears to be unravelling: civility seems like an old-fashioned habit, honesty like an optional exercise and trust like the relic of another time. Some observersWhy have so many different people in so many different times and places been convinced that their fellow citizens are now less moral than they once were? One possibility is that morality has, in fact, been declining worldwide for millennia—declining so steadily and so precipitously that people in every era have been able to observe that decline in the brief span of a human lifetime. The other possibility is that the perception of moral decline is a psychological illusion to which people all over the world and throughout history have been susceptible. We provide evidence for the latter possibility. First, we show that people in at least 60 nations do indeed believe that morality is declining, and that they have believed this for at least 70 years. Second, we show that people attribute this decline both to the decreasing morality of individuals as they age and to the decreasing morality of successive generations. Third, we show that people’s reports of the current morality of their contemporaries have not declined over time, which strongly suggests that the perception of moral decline is an illusion. Fourth and finally, we describe tests of a simple psychological mechanism that can produce the illusion of moral decline and can predict some of the circumstances under which it will be attenuated, eliminated or reversed .8, which ranges from the altruistic9 to the barbaric10. But like most social observers, Livy was not remarking on the moral extremes—on the rare heroic deed or occasional heinous crime that few people ever perform or experience. Rather, he was remarking on the ways in which ordinary people behave in their daily lives. Do modern people, like Livy, believe that their contemporaries are less honest and kind than they used to be? Do they think their neighbours are less generous and less helpful, that their co-workers are more likely to treat each other disrespectfully and betray each other’s trust? Survey researchers have been asking people about their perceptions of changes in these everyday moral qualities since at least 1949, but the full corpus of relevant survey data has never been systematically assembled and analysed. We began by doing that.Morality refers primarily to people’s treatment of each otherb = 0.07, 95% confidence interval (CI) = , t(175) = 0.77, P = 0.45, adjusted R2 = −0.002, and the same model fit in a Bayesian framework indicated strong evidence of no effect (Bayes Factor of 0.04), which is to say that US Americans have been reporting moral decline at the same rate for as long as researchers have been asking them about it. .In study 1, we searched the databases of major survey research providers  = 2.42, P = 0.02, adjusted R2 = 0.10, which is precisely what one would expect if participants believed that morality has been declining continuously. Second, participants reported increases in morality when asked about a few specific issues on which social progress has clearly been made: for example, 59% of participants reported improved treatment of African Americans, 51% reported improved treatment of people with physical disabilities and 50% reported improved treatment of gay people. The fact that participants calculated moral decline cumulatively across time periods and acknowledged special exceptions to the general trend suggests that they were reporting well-considered beliefs, and not merely expressing some vague sense of despair about humanity. Indeed, in the Supplementary Two more findings were noteworthy. First, participants in study 1 were more likely to perceive moral decline when they were asked about longer periods of time  than about shorter periods of time (“the last year”), 11) and 2006 (ref. 12) and, as Fig. The perception of moral decline was not unique to US Americans. We resampled the databases of major survey research providers and found 58 survey items that asked a total of 354,120 participants in 59 nations other than the United States if and how they thought other people’s morality had changed over time , study 2b  and study 2c  perceived moral decline. Specifically, in study 2a, participants rated people as less kind, honest, nice and good in 2020 (mean (M) = 4.39) than in 2010  = −9.38, P < 0.001, Cohen’s d = −0.50), or in 2000  = −13.23, P < 0.001, d = −0.71), and as less kind, honest, nice and good in 2010 than in 2000, b = −0.15, 95% CI = , t(1394) = −3.85, P < 0.001, d = −0.21. We also conducted two direct replications of study 2a , as well as in various other years that differed by study. Methodological details can be found in the concluding method section and in the M = 4.28) than in 2016  = −2.87, P = 0.047, d = −0.33), in 2014  = −3.14; P = 0.02, d = −0.37), in 2012: M = 4.59, b = −0.30, 95% CI = , t(735) = −4.16, P < 0.001, d = −0.48) and in 2010 M = 4.66, b = −0.37, 95% CI = , t(735) = −5.08, P < 0.001, d = −0.59). Participants in study 2b also rated people in 2018 as less kind, honest, nice and good than in people in 2010  = −3.61, P = 0.004, d = −0.32). No other comparisons were significant .In study 2b, participants rated people as less kind, honest, nice and good in 2020 (M = 4.28) than in the year the participant turned 20 years old  = −9.21, P < 0.001, d = −0.72) and in the year the participant was born  = −14.27, P < 0.001, d = −1.08). Participants also rated people as less kind, honest, nice and good in the year the participant turned 20 years old than in the year the participant was born, b = −0.31, 95% CI = , t(675) = −4.70, P < 0.001, d = −0.37.In study 2c, participants rated people as less kind, honest, nice and good in 2020  = −4.03, P < 0.001. No other effects in study 2a were significant . An additional exploratory one-sample t-test indicated that, although more conservative participants perceived more moral decline than did more liberal participants, more liberal participants perceived moral decline as well, M = −0.35, 95% CI = , t(398) = −4.96, d = 0.28, P < 0.001, d = 0.28. In study 2b, no other effects were significant . In study 2c, more conservative participants perceived more moral decline than did more liberal participants, b = −0.15, 95% CI = , t(329) = −2.15, P = 0.03, but more liberal participants perceived moral decline as well, M = −0.80, 95% CI = , t(159) = −7.23, P < 0.001, d = 0.57.In studies 2a–c, we also examined the effects of age, gender, race, education , political ideology  and parental status  on perceptions of moral decline using an exploratory linear regression. In study 2a, more conservative participants perceived more decline, b = −0.02, 95% CI = , t(329) = −2.61, P = 0.01. No other effects were significant . We further investigated the effect of age in study 2c by creating two moral decline scores: specifically, (1) we subtracted participants’ ratings of people in the year the participant was 20 years old from their ratings of people in 2020 and (2) we subtracted participants’ ratings of people in the year the participant was born from their ratings of people in 2020. An exploratory linear model indicated that older participants perceived more moral decline than did younger participants both compared to the year in which they turned 20 years old: b = −0.02, t(320) = −3.30, 95% CI = , P < 0.001, and compared to the year in which they were born: b = −0.02, t(345) = −3.77, 95% CI = , P < 0.001. Did older participants perceive more moral decline simply because they were considering longer periods of time? Yes. We created a measure of the annual rate of moral decline by subtracting the participants’ ratings of people in the year the participant was born from their rating of people in 2020, and then dividing that value by the participant’s age. We then fit an exploratory linear model with the perceived annual rate of moral decline as the outcome and age as a predictor. The main effect of age was not significant, b = −0.0002, 95% CI = , t(345) = −1.05, P = 0.29, and refitting the same model in a Bayesian framework provided strong evidence that perceived moral decline per year did not differ by age  in the region of practical equivalence (ROPE)). In other words, younger and older participants did not report different annual rates of moral decline, which is to say that they reported different total amounts of moral decline only because they were reporting on moral decline across different numbers of years.In study 2c, older participants perceived more moral decline than did younger participants, T1 and T2) because (1) individuals who are moral at T1 are less moral when they reach T2 , and/or (2) older people who were alive at T1 but who died before T2 are more moral than younger people who were alive at T2 but who were not yet born (or who were not yet adults and therefore not sampled) at T1 . When the average morality of a population declines over very short time periods , the decline is probably the result of personal change , and when the average morality of a population declines over very long time periods , the decline is necessarily the result of interpersonal replacement .People clearly perceive moral decline, but to what do they attribute it? There are two possibilities. The average morality of a population may decline between two points in time , we asked a sample of US Americans to rate how “kind, honest, nice, and good” people were in 2020 (the year the study was conducted) and in 2005. Methodological details can be found in the So, what about moral decline over the intermediate time periods that participants in studies 2a–c were asked about? To which of these sources—personal change or interpersonal replacement—do people attribute such decline? In study 3  than in 2005 (M = 4.89), t(318) = −9.88, 95% CI = , d = 0.55, P < 0.001. To determine whether participants attributed this moral decline to personal change and/or to interpersonal replacement, we used linear regression to determine whether and how well participants’ perceptions of personal change and of interpersonal replacement predicted their perceptions of moral decline. Both measures significantly predicted participants’ perceptions of moral decline  = 9.96, P < 0.001; interpersonal replacement b = 0.17, t(316) = 6.52, 95% CI = , P < 0.001; adjusted R2 = 0.36). We refit the model to include age, gender, race, political ideology, education and parental status as covariates, and the effects of personal change and interpersonal replacement both remained significant  = 9.87, P < 0.001; interpersonal replacement b = 0.18, 95% CI = , t(304) = 6.71, P < 0.001; adjusted R2 = 0.38).A paired samples 14. The fact that people attribute moral decline to both sources may help explain why their perceptions of moral decline are so robust, appearing in study 3, in the archival data of study 1 and in the original data collected for studies 2a–c.In short, participants in study 3 believed that morality had declined on average over a 15-year period, and they attributed that decline both to the decreasing morality of individuals over time and to the decreasing morality of successive generations16. On average, modern humans treat each other far better than their forebears ever did—which is not what one would expect if honesty, kindness, niceness and goodness had been decreasing steadily, year after year, for millennia. Although there are no similarly objective historical records of everyday morality—of how often people offer their seats to an elderly person, give directions to a lost tourist or help their neighbour fix a fence—there are subjective measures of such things.People believe that morality is declining. Is it? Societies keep (or at least leave) reasonably good records of extremely immoral behaviour such as slaughter and conquest, slavery and subjugation or murder and rape, and careful analyses of those historical records strongly suggest that these objective indicators of immorality have decreased significantly over the last few centuriesRecall that in study 1, we examined people’s reports of moral change, which were obtained when survey researchers asked people to mentally compare the morality of people in the present to the morality of people at some point in the past and then report the direction of the difference. But, for decades, survey researchers have also been asking people to report directly on the moral values, traits and behaviours of themselves and their contemporaries in the present: “Were you treated with respect all day yesterday?” or “Would you say that most of the time people try to be helpful, or that they are mostly just looking out for themselves?” or “During the past 12 months, how often have you carried a stranger’s belongings, like groceries, a suitcase, or shopping bag?”  and found similar results: on average, the year in which the survey was conducted explained less than 0.2% of the variance in responses  mechanism comports well with the results of the studies we have described, but it also makes at least two testable predictions. Specifically, the BEAM mechanism predicts that the illusion of moral decline should be attenuated, eliminated or even reversed when (1) people are exposed to a disproportionate amount of positive rather than negative information about the moral behaviour of others, as they are with their families, friends and associates, and (2) when people are asked about times for which they have little or no information in memory, such as in the years before they were born. In the Supplementary n = 283 respondents on Amazon Mechanical Turk) tested the hypothesis that the illusion of moral decline is attenuated, eliminated or reversed when participants are asked to rate people in their personal worlds rather than people in general. As described in the Study 5a , t(282) = −6.04, 95% CI = , d = 0.36, P < 0.001. Second, participants believed that individuals in 2020 were not as kind, honest, nice and good as those same individuals had been in 2005, M = −0.15, t(282) = −2.67, 95% CI = , d = 0.16, P = 0.008, and that younger people in 2020 were not as kind, honest, nice and good as older people were in 2005, M = −0.44, t(282) = −5.82, 95% CI = , d = 0.35, P < 0.001. In other words, as in study 3, participants believed that morality had declined among individuals and between successive generations. These results are illustrated in Fig. We used one-sample 5 M = −0.6, t(282)M = 0.23, t(255) = 4.86, 95% CI = , d = 0.30, P < 0.001. Second, although participants believed that the younger people who were in their personal worlds in 2020 (but not in 2005) were not as kind, honest, nice and good as the older people who were in their personal worlds in 2005 (but not in 2020), M = −0.23, t(144) = −3.23, 95% CI = , d = 0.27, P = 0.002, this difference was smaller among people in their personal worlds than it was among people in general, t(144) = −2.56, 95% CI = , d = 0.18, P = 0.01.Did participants believe the same things about people in their personal worlds? No. First, participants believed that the individuals who were in their personal worlds in both 2005 and 2020 had shown moral improvement over that period rather than moral decline, b = −0.02, 95% CI = , t(272) = −2.93, P = 0.004, and non-parents perceived more moral decline than did parents, b = 0.29, 95% CI = , t(271) = 2.12, P = 0.03, adjusted R2 = 0.07. No other effects were significant .To investigate the effects of demographic variables on the perception of moral decline, we fit the same exploratory model used in studies 2a–c. The outcome variable was participants’ perceptions of moral decline between 2005 and 2020. Older participants perceived more moral decline than did younger participants, In short, participants in study 5a believed that morality had declined among people in general, but this effect was reversed  or attenuated  among the people they personally knew. We hasten to note that there are surely many reasons why people might think differently about people in their personal worlds than about people in general and that the BEAM mechanism is, at best, just one.n = 387 respondents on Amazon Mechanical Turk) tested the hypothesis that the illusion of moral decline is attenuated, eliminated or reversed when participants are asked to rate the morality of people in general in the years before the participant was born. Participants rated how kind, honest, nice and good people in general are or were at four points in time: in the current year (which was 2021), 20 years after the participant was born, the year the participant was born, 20 years before the participant was born and 40 years before the participant was born. We fit the same model and planned contrasts used in studies 2a–c. As in our previous studies, participants perceived moral decline among people in general in the years after the participant was born. Specifically, participants believed that people in general were (1) less kind, honest, nice and good in 2021 (M = 4.27) than they were in the year the participant was 20 years old (M = 4.96), b = –0.68, 95% CI = , t(1513) = –10.07, P < 0.001, d = −0.75 and (2) less kind, honest, nice and good in the year the participant was 20 years old than they were in the year the participant was born (M = 5.13), b = –0.18, 95% CI , t(1513) = –2.60, P = 0.03, d = −0.19. However, there was no evidence to suggest that participants perceived moral decline in the years before they were born. Specifically, there was no evidence that participants believed that people in general were (1) any more or less kind, honest, nice and good in the year the participant was born (M = 5.13) than they were 20 years before the participant was born (M = 5.14), b = –0.01, 95% CI = , t(1506) = –0.16, P = 0.87, d = −0.01 and (2) any more or less kind, honest, nice and good 20 years before the participant was born (M = 5.14) than they were 40 years before the participant was born (M = 5.05), b = 0.09, 95% CI = , t(1506) = −1.42, P = 0.31, d = 0.10. Equivalence tests using the ‘parameters’ package in R26 indicated that there was insufficient evidence to conclude that participants’ ratings for 40 years before their birth and 20 years before their birth were equivalent , but that there was sufficient evidence to conclude that participants’ ratings for 20 years before their birth and the year of their birth were equivalent . In short, participants believed that moral decline began at about roughly the same time they appeared on Earth. These results are illustrated in Fig. Study 5b  = −5.81, P < 0.001, but a one-sample t-test indicated that more liberal participants perceived moral decline as well, b = −0.46, 95% CI = , t(196) = −5.11, P < 0.001, d = 0.36. Although older participants perceived more moral decline than did younger participants, b = −0.01, 95% CI = , t(374) = −2.60, P = 0.009, this was because older participants were perceiving moral decline over a longer period of time. Indeed, the same analysis used in study 2c indicated no evidence that older and younger participants perceived different annual rates of moral decline between the year they were born and 2021, b = 0.000005, 95% CI = , t(385) = 0.04, P = 0.97, consistent with the results of study 2c.To investigate the effects of demographic variables on the perception of moral decline, we fit the same exploratory model used in studies 2a–c. The outcome variable was perceived moral decline between the year of the participant’s birth and 2021. More conservative participants perceived more moral decline than did more liberal participants, Studies 5a and 5b show that when participants were asked to assess the morality of people about whom they had mainly positive information in memory  or about whom they had little or no information in memory , the perception of moral decline was attenuated, eliminated or reversed, just as the BEAM mechanism predicts. The illusion of moral decline is a robust phenomenon that surely has several causes, and no one can say which of them produced the illusion that our studies have documented. Studies 5a and 5b do not directly implicate the BEAM mechanism in that production but they do make it a viable candidate for future research.Participants in the foregoing studies believed that morality has declined, and they believed this in every decade and in every nation we studied. They believed the decline began somewhere around the time they were born, regardless of when that was, and they believed it continues to this day. They believed the decline was a result both of individuals becoming less moral as they move through time and of the replacement of more moral people by less moral people. And they believed that the people they personally know and the people who lived before they did are exceptions to this rule. About all these things, they were almost certainly mistaken. One reason they may have held these mistaken beliefs is that they may typically have encountered more negative than positive information about the morality of contemporaries whom they did not personally know, and the negative information may have faded more quickly from memory or lost its emotional impact more quickly than the positive information did, leading them to believe that people today are not as kind, nice, honest or good as once upon a time they were.Like all studies, ours have limitations. For example, studies 1 and 4 made use of archival data that were not collected for the purposes to which we put them and that were therefore less than ideal. For example, some of the items we analysed asked participants for their perceptions of changes in ‘moral values’ without specifying what those values were, some failed to specify the time in the past to which the present was to be compared, and some contained ambiguous wording that was not optimal for extracting accurate measures of people’s perceptions of moral decline. Moreover, all the items asked participants about the presence or absence of moral decline rather than asking them to rate the level of morality of people in both the present and the past. These limitations were addressed by studies 2a–c, but these studies had limitations of their own . And although studies 5a–b demonstrated the viability of the BEAM mechanism, they do not tell us whether it was the cause of the illusion of moral decline that our other studies documented.27. The United States faces many well-documented problems, from climate change and terrorism to racial injustice and economic inequality—and yet, most US Americans believe their government should devote scarce resources to reversing an imaginary trend. The belief that everyday morality is on the wane may also affect people’s interpersonal behaviour. For example, research shows that people are reluctant to seek the aid and comfort of those whom they do not know because they underestimate how willingly those people would provide it29. The illusion of moral decline may be one of the reasons people do not depend as much as they might on the kindness of strangers—an act that might well ameliorate the illusion itself. The illusion of moral decline may also leave people dangerously susceptible to manipulation by bad actors. Research shows that people are especially influenced by ‘dynamic norms’, which are perceived changes in customary ways of behaving5. If low morality is a cause for concern, then declining morality may be a veritable call to arms, and leaders who promise to halt that illusory slide—to “make America great again”, as it were—may have outsized appeal. Our studies indicate that the perception of moral decline is pervasive, perdurable, unfounded and easily produced. Achieving a better understanding of this phenomenon would seem a timely task.With that said, the illusion of moral decline seems to be a robust phenomenon that may have troubling consequences. For example, in 2015, 76% of US Americans agreed that “addressing the moral breakdown of the country” should be a high priority for their governmentIn study 1, we conducted keyword-term searches of the Roper Center for Public Opinion iPoll Database, and manually searched the databases of the General Social Survey, Pew Research Center, Gallup, the American National Election Studies, the World Values Survey, the European Social Survey and the European Values Survey to locate survey items that asked participants if and how they thought other people’s morality had changed over time. In our analyses, we included all surveys that (1) used a representative sample of US American participants, and (2) explicitly asked participants about their perceptions of changes in values, traits and behaviours that have traditionally been taken as indicators of morality by a wide range of US Americans . We excluded from our analyses items that asked participants about their perceptions of special topics whose moral relevance either changed considerably over time  or differed substantially across members of the population . We also excluded items that asked participants about the morality of special subpopulations  rather than about all US Americans or about people in general. Further information, including search terms and all survey items included in study 1, can be found in the All original data collection in this and subsequent studies followed all ethical regulations and was approved by the Institutional Review Board of Harvard University.Mage = 45.74 years, 73% white, 13% Black, 7% Asian, 4% Hispanic, 1% American Indian or Alaska Native, 1% other, 2% ‘more than one of the above’) were paid US$0.75 each for their participation.We recruited a nationally representative sample of US American adults using Prolific, an online sample provider. This sample was constructed to represent the US American adult population in terms of gender, race and age. Because we did not know the size of the effect we were studying, we sought to make our sample comparable in size to the samples in study 1 by recruiting 1,000 participants. Nine-hundred and ninety-nine people . These exclusions do not meaningfully affect the results.One hundred and eighty-one participants failed the attention check embedded in the demographics and were excluded from all analyses. Another 120 participants gave answers to the consistency check question that were inconsistent with their previous answers; they were also excluded. This left 698 participants in all analyses . The model included a fixed effect of the year of each rating and a random intercept for each participant. For this and all models, we checked model assumptions by plotting the outcome variable, residuals and fitted values. All tests we report are two-tailed.To analyse the data, we fit a linear mixed effects model using the lme4 package in Rd = 0.30 or larger, reasoning that this would be sufficient to detect effects similar to the effect we detected in Study 2a. Two-hundred and thirty-six people responded to an advertisement for a study on Amazon Mechanical Turk. To participate, respondents had to pass a three-item test that required them to know that (1) children in kindergarten are 3 or 4 years old, (2) a US American ZIP code is a series of five digits and (3) eating turkey is not associated with Halloween. Thirty-six respondents answered at least one of these three questions incorrectly and were not allowed to participate. The remaining 200 respondents  were allowed to participate in the study in exchange for US$0.75.We powered study 2b to detect an effect of After providing informed consent, participants followed study 2a’s procedure except they were asked about different years. Specifically, participants were first asked, “How kind, honest, nice, and good are people today?” and were then asked the same question for “two years ago”, “four years ago”, “six years ago”, “eight years ago” and “ten years ago”, in that order. All questions were answered using a seven-point Likert scale with endpoints labelled ‘not very’ and ‘very’. As a consistency check, participants then answered the following question: “When it comes to being kind, honest, nice, and good—are people more so today compared to ten years ago, less so today compared to ten years ago, or the same?” Participants were then asked to explain their answer in an open-ended question. Finally, participants were asked some demographic questions, as well as an attention check question that required them to select the option ‘other’ and to type the word ‘day’. Participants were compensated and dismissed.Mage = 36.59 years, 75% white, 9% Black, 7% Hispanic, 5% Asian, 1% Hawaiian or Pacific Islander, 3% more the one of the above). These exclusions only meaningfully affect the results in one case, namely, that when all participants are included, the difference between 2020 and 2016 is not significant.Fifteen participants failed the attention check, and a further 37 participants failed the consistency check by giving an answer that was inconsistent with their scale ratings. The data from these participants were excluded from all analyses, leaving 148 participants .We sought to recruit a sample of people who varied widely in terms of age. As such, we created a survey with a quota of 50 participants in each of the following age groups: 18–24, 25–29, 30–34, 35–39, 40–44, 45–49, 50–54, 55–59, 60–64 and 65–69 years. This sample size gave us sufficient power to detect the effects we had detected in studies 2a and 2b. Respondents selected their age group on accessing the study, and once the quota for a group was reached, further respondents from that group were not allowed to participate. Respondents younger than 18 or older than 69 were not allowed to participate.Mage = 41.27 years, 72% white, 15% Black, 7% Asian, 4% Hispanic, 1% American Indian or Alaska Native, 2% more than one of the above) were allowed to participate in the study in exchange for US$0.75.Respondents responded to an advertisement for a study on Amazon Mechanical Turk. Respondents who accessed the survey before the quota for their age group was reached were asked to complete a three-item test of English proficiency and knowledge of US American culture. Specifically, they were required to demonstrate that they knew that (1) bell bottoms are not a type of footwear, (2) an RSVP is a required response to a wedding invitation and (3) a sign reading ‘out of order’ is best paired with an elevator. Three hundred and one respondents answered one or more of these questions incorrectly and were not allowed to participate. The remaining 484 respondents  were about 20 years old, and at about the time they (the participants) were born. This was done by adjusting the wording of the subsequent questions on the basis of the participant’s age. For example, if the participant was between 30 and 34 years old, they were asked “How kind, honest, nice, and good were people about ten years ago?” and then “How kind, honest, nice, and good were people about 30 years ago?” If participants were under 25 years, they answered only the questions for today and when they were born. All questions were answered using a seven-point Likert scale with endpoints labelled ‘not very’ and ‘very’. As in previous studies, participants were then given a consistency check that required them to remember whether they had rated people today as more, equally or less moral compared to people in the year they were born. Participants then answered some further exploratory and demographic questions. Embedded among them was an attention check that required participants to select the option ‘other’ and type the word ‘apple’. Finally, participants were compensated and dismissed.Mage = 42.57 years, 78% white, 9% Black, 7% Asian, 4% Hispanic, 2% ‘more than one of the above’) were included in all analyses. These exclusions do not meaningfully change the results.Twenty-eight participants failed the attention check and their data were excluded from all analyses. Seventy-three more participants reported an age at the end of the study that was inconsistent with the age group they selected at the beginning of the study and the data from these participants were also excluded from all analyses. An extra 64 participants failed the consistency check and data from these participants were also excluded from all analyses. The data from the remaining 347 participants .Mage = 40.42 years, 77% white, 9% Black, 7% Asian, 5% Hispanic, 1% ‘more than one of the above’) provided informed consent and became participants in the study in exchange for US$0.75.Respondents responded to an advertisement for a study on Amazon Mechanical Turk. As in study 2c, we sought to recruit a sample of people who varied widely in terms of age and that was large enough to provide sufficient power to detect the effects we had detected in studies 2a and 2b. We created a survey with quota of 150 for each of three age groups: 20–34, 35–49 and 50–64. Anyone younger than 20 or older than 64 was not allowed to participate. Respondents were asked to complete the same test of English language and US American culture as in study 2c. Four hundred and forty-four respondents  and also “about 15 years ago” (about 2005) on seven-point Likert scales with endpoints labelled ‘not very’ and ‘very’ and then completed a consistency check that asked them to recall the answers they had just given. The difference between these two ratings was used as a measure of participants’ perception of moral decline between 2005 and 2020. Participants then answered the following questions using the same seven-point Likert scales: “How kind, honest, nice, and good are people who are currently between the ages of 35 and 95?”; “How kind, honest, nice, and good are people who are currently between the ages of 20 and 35?”; “Thinking again of people who are currently between the ages of 35 and 95, how kind, honest, nice, and good were they about 15 years ago?” and “About 15 years ago, how kind, honest, nice, and good were people who were then between the ages of 80 and 95?” Participants then answered some demographics questions, among which was embedded an ‘attention check question’ that instructed participants to select the option ‘other’ and to type the word ‘cloud’. Finally, participants were compensated and dismissed.Mage = 41.02, 77% white, 8% Black, 8% Asian, 5% Hispanic, 1% more than one of the above). These exclusions do not meaningfully affect the results.Forty-eight participants failed the attention check, and a further 15 participants reported an age at the end of the study that was inconsistent with the age group they reported at the beginning of the study. An extra 77 participants failed the consistency check. The data from all of these participants were excluded from all analyses, leaving 319 participants  from ratings of 35–95-year olds in the present (2020). We created an interpersonal replacement score by subtracting ratings of 80–95-year olds about 15 years ago (in 2005) from ratings of 20–35-year olds in the present (2020). The descriptive statistics for people in general and each of the subgroups about which participants were asked are shown in Extended Data Fig. Using a standard linear model, we entered participants’ personal change and cohort replacement scores as predictors, and the outcome was participants’ overall perception of moral decline between 2005 and 2020.In study 4, we conducted keyword-term searches of the Roper Center for Public Opinion iPoll Database .We fit a linear model for each survey. The year of each survey was always entered as a predictor and the outcome was always the average perception of current morality. We used As in study 2c, we sought to recruit a sample of people who varied widely in terms of age and that was large enough to provide sufficient power to detect the effects we had detected in previous studies. We created a survey with a quota of 50 participants in each of three age groups: 20–34, 35–49 and 50–64 years. Anyone who was either younger than 20 years or older than 64 years was not allowed to participate.Mage = 37.74 years, 65% white, 24% Black, 7% Asian, 2% Hispanic, 1% American Indian or Alaska Native, 1% more than one of the above) provided informed consent and became participants in the study in exchange for US$0.75.One thousand and twenty-one people responded to an advertisement for a study on Amazon Mechanical Turk. They completed the same test of English language and US American culture as in study 2c. Five hundred and twenty-one respondents answered at least one of the questions incorrectly and were not allowed to participate. The remaining 500 respondents  were included in all analyses. These exclusions affect the results in a few cases. Specifically, when excluded participants are included, the overall perception of moral decline and personal change for people in general are not significant. All other effects remain significant.One hundred and seventy-nine participants failed the first attention check, and another 21 failed the second attention check. Another 15 participants reported an age at the end of the study that was inconsistent with the birth year they reported at the beginning. The data from all these participants were excluded from all analyses. The remaining 283 participants .Mage = 43.96 years, 78% white, 10% Asian, 5% Black, 4% Hispanic, 3% more than one of the above) were allowed to participate in the study in exchange for US$0.75.Because this study was a replication and extension of study 2c, we sought to collect a similar sample size to have the power to detect similar effects, and we used the same age quotas as in Study 2c. One thousand eighty-two people responded to an advertisement for a study on Amazon Mechanical Turk. Twenty-one of these opened the study but did not complete it. Five hundred and sixty people responded after the quota for their age group had been reached and were not allowed to participate in the study. Respondents who responded before the quota for their age group was reached completed the same three-item test of US American culture and English language used in study 2c. Twenty-three respondents answered one or more of these questions incorrectly and were not allowed to participate in the study. The remaining 499 respondents  were included in all analyses. These exclusions affect the results in one case: when excluded participants are included, participants perceived moral improvement from 40 years before birth to 20 years before birth. All other effects remain the same.Forty-four participants failed the attention check and their data were excluded from all analyses. Seven more participants reported an age at the end of the study that was inconsistent with the age group they selected at the beginning of the study and their data were also excluded from all analyses. Sixty-one more participants failed the consistency check and their data were also excluded from all analyses. The data from the remaining 387 participants .Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-06137-x.Supplementary InformationReporting Summary


SGN were isolated from neonatal rats (P 2–5) and cultured in serum-free medium. EPO and Cometin of mouse and human origin were added in concentrations of 0.1, 1, and 10 ng/mL and 0.1, 1, and 10 μg/mL, respectively. The SGN survival rate and morphology, and the neurite outgrowth were determined and compared to negative (no additives) and positive  controls. A neuroprotective effect of 10 μg/mL human Cometin comparable to that obtained with BDNF was observed in the SGN-culture. In contrast, mouse Cometin was ineffective. A similar influence of 10 μg/mL human and mouse and 1 μg/mL human Cometin on the length of regenerated neurites compared to BDNF was also detected. No other Cometin-conditions, and none of the EPO-conditions tested had neuroprotective or neuritogenic effects or influenced the neuronal morphology of the SGN. The neuroprotective effect of 10 μg/mL human Cometin on SGN indicates it is a potentially interesting protein for the supportive treatment of inner ear disorders. The finding that mouse Cometin had no effect on the SGN in the parallel-performed experiments underlines the importance of species origin of molecules being screened for therapeutic purpose.Neurotrophic factors (NTF) play key roles in the survival of neurons, making them promising candidates for therapy of neurodegenerative diseases. In the case of the inner ear, sensorineural hearing loss (SNHL) is characterized over time by a degeneration of the primary auditory neurons, the spiral ganglion neurons (SGN). It is well known that selected NTF can protect SGN from degeneration, which positively influences the outcome of cochlear implants, the treatment of choice for patients with profound to severe SNHL. However, the outcome of studies investigating protective effects of NTF on auditory neurons are in some cases of high variability. We hypothesize that the factor origin may be one aspect that affects the neuroprotective potential. The aim of this study was to investigate the neuroprotective potential of  It is estimated that by 2050 over 900 million people will have disabling hearing loss . Sensoriin vivo .per se (For Erythropoietin (EPO) neuroprotective effects in preclinical models of acute and chronic degenerative disorders  and imprper se  have alsper se . For SGNper se , (ii) reper se , (iii) hper se  or (iv) per se . Additioin vitro. In addition to EPO we also tested the SGN supportive effects of Cometin  obtained from different species. Cometin is a secreted neurotrophic protein with tissue specific expression  .All experiments were performed in accordance with the German “Law on Protecting Animals” and with the European Directive 86/609/EEC of the European Communities Council for protection of animals used for experimental purposes and registered with the local authorities of Hannover Medical School. Due to its common use as erformed . Neonataerformed . In shorThe enzymatic and mechanical dissociation of the SGC was performed using 0.1% trypsin  and 0.01% DNase I . Enzymatic dissociation was stopped by withdrawal of the supernatant and addition of fetal calf serum  and the SGC were mechanically dissociated in 1 mL of the serum-free medium (see below). Trituration was repeated until no cell clusters were visible in the suspension. The cell number was counted using the trypan blue  exclusion test in a Neubauer chamber . Finally, the cells were seeded in poly-DL-ornithine /laminin  coated 96-multiwell culture plates .2.2.4 cells/well. The serum-free culture medium consisted of Panserin 401  supplemented with Hepes-buffer solution , PBS , penicillin , glucose , insulin  and N2-supplement . Culture was performed in an incubator  in a humidified atmosphere with 5% CO2 at 37°C.Cells were seeded in a concentration of 1 × 10E. coli; Invitrogen) served as positive control (PC)  , 2020, w2.3.Cells were immunocytochemically stained with monoclonal antibody against Neurofilament 200 kD to distinguish neurons from the non-neuronal cells. A detailed protocol has been described previously . Briefly2.4.2.4.1.Surviving SGN were defined as cells stained positive for neurofilament and with neurites at least three times longer than the soma diameter  and were2.4.2.According to 2.4.3.For the analysis of the SGN neurite length, images were taken from at least five fields of view evenly distributed over the well area  and proc2.5.p values less than 0.05.Statistical analysis was performed using GraphPad Prism® software 9. Data were checked for normal distribution using the D’Agostino and Pearson normality test. Data from neuronal survival rate and morphology passed normality test and were analyzed using one-way ANOVA followed by Tukey’s Multiple Comparison Test. The measured neurite length were not normally distributed and Kruskal Wallis test followed by Dunn’s Multiple Comparison Test was performed. The data are reported as mean ± standard error of the mean (SEM). Statistical significance was considered at 3.3.1.p < 0.001; EPO: p < 0.01).p < 0.001), the lower concentrations of 1 μg/mL and 0.1 μg/mL human Cometin  and the 10 μg/mL, 1 μg/mL, and 0.1 μg/mL concentrations of mouse Cometin . Compared to the PC, 10 μg/mL human Cometin performed as well as BDNF in supporting the neuronal survival of SGN. With the exception of 10 μg/mL human Cometin, none of the lower human or any of the mouse Cometin concentrations had a significant effect on the neuronal survival compared to the NC, and survival was significantly lower than in the PC. A within species comparison indicated that only 10 μg/mL human Cometin performed better than the equivalent mouse concentration. This was also the case for comparison of the tested three concentrations of human and mouse Cometin.The addition of 10 μg/mL human Cometin resulted in the highest detected survival rate of SGN compared to all other conditions. This increase in SGN survival was significant compared to the NC (p < 0.001), PC (p < 0.001), and to all other EPO conditions  occurred after the addition of 10 ng/mL human EPO. All added EPO concentrations, whether human or mouse, resulted in significantly lower survival rates than that observed in the PC. Overall, a comparison between the two species and the three concentrations within species revealed that only the 10 ng/mL concentration of human EPO significantly reduced SGN survival.In the EPO experiments, none of the tested mouse concentrations had a significant effect on SGN survival when compared to NC. Human EPO did not differ from NC when adding 0.1 ng/mL or 1 ng/mL to the SGN. Moreover, the lowest detected survival rate compared to NC  in Cometin and EPO conditions. Therefore, only the percentage of monopolar, bipolar and no neurite neurons are presented in 3.3.Measured neurite lengths of Cometin and EPO experiments are illustrated in p < 0.05) and 0.1 μg/mL human and mouse Cometin . For 10 μg/mL human and mouse Cometin and 1 μg/mL human Cometin, neurite length did not differ significantly from PC. Overall, the shortest neurites were measured for 0.1 μg/mL human Cometin. This length was significantly shorter than in PC and for10 μg/ml human Cometin . Except for 0.1 μg/mL human Cometin, no differences were observed within the mouse and human Cometin groups or between the three tested concentrations.Cometin treatment had no significant effect on the length of outgrowing neurites compared to NC, regardless of species origin or concentration. Neurites were significantly shorter than in the PC for SGN treated with 1 μg/mL mouse Cometin (MC1: p < 0.001). Compared to PC, all EPO-treated groups resulted in significantly shorter neurites, regardless of species source and concentration . Neurite outgrowth was overall significantly shortest for 10 ng/mL human EPO  with the exception of 1 ng/mL mouse EPO. Within the human EPO concentrations tested, 10 ng/mL resulted in the shortest neurites, while there was no difference in neurite length between 1 ng/mL and 0.1 ng/mL EPO. The three tested concentrations of mouse EPO had no significant different effect on the neurite length. Comparing human with mouse EPO at the same concentrations, neurites were longer for 10 ng/mL mouse EPO (ME10: p < 0.05), while in case of 1 ng/mL neurites were longer for human EPO (ME1: p < 0.001). There was no detectable difference between species for the 0.1 ng/mL concentration.In the EPO experiments, neurite outgrowth was significantly shorter for neurons treated with the different concentrations of mouse EPO when compared to NC . SGN provided with 1 ng/mL or 0.1 ng/mL human EPO regenerated neurites in a length not differing significantly from NC, while 10 ng/mL human EPO induced significantly shorter neurite outgrowth  under Germany’s Excellence Strategy – EXC 2177/1–Project ID 390895286. This publication is funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the “Open Access Publikationskosten” program.Authors GM and KP were employed by company Hoba Therapeutics ApS.All authors declare that this study received funding from Hoba Therapeutics ApS. This funder had the following involvement in the study: contribution of reagents and materials, study design, and preparation of the manuscript.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.