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We summarize the copy number variations (CNVs) and phenotype spectrum of infantile epileptic spasms syndrome (IESS) in a Chinese cohort. The CNVs were identified by genomic copy number variation sequencing. The CNVs and clinical data were analyzed. 74 IESS children with CNVs were enrolled. 35 kinds of CNVs were identified. There were 11 deletions and 5 duplications not reported previously in IESS, including 2 CNVs not reported in epilepsy. 87.8% were de novo, 9.5% were inherited from mother and 2.7% from father. Mosaicism occurred in one patient with Xq21.31q25 duplication. 16.2% (12/74) were 1p36 deletion, and 20.3% (15/74) were 15q11-q13 duplication. The age of seizure onset ranged from 17 days to 24 months. Seizure types included epileptic spasms, focal seizures, tonic seizures, and myoclonic seizures. All patients displayed developmental delay. Additional features included craniofacial anomaly, microcephaly, congenital heart defects, and hemangioma. 29.7% of patients were seizure-free for more than 12 months, and 70.3% still had seizures after trying 2 or more anti-seizure medications. In conclusion, CNVs is a prominent etiology of IESS. 1p36 deletion and 15q duplication occurred most frequently. CNV detection should be performed in patients with IESS of unknown causes, especially in children with craniofacial anomalies and microcephaly.
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Variaciones en el Número de Copia de ADN , Fenotipo , Espasmos Infantiles , Humanos , Variaciones en el Número de Copia de ADN/genética , Espasmos Infantiles/genética , Femenino , Masculino , Lactante , Duplicación Cromosómica/genética , Cromosomas Humanos Par 15/genética , Preescolar , Recién Nacido , Deleción Cromosómica , Mosaicismo , Aberraciones Cromosómicas , Discapacidad IntelectualRESUMEN
BACKGROUND: There is limited evidence on the association between housing debt and depressive symptoms in China. This study aimed to examine the impact of housing debt on depressive symptoms and explore the heterogeneous impacts arising from two sources of housing debt and two types of housing demands. METHODS: Using data from the 2016 and 2018 China Family Panel Studies (CFPS), this study included 25,232 Chinese individuals. Depressive symptoms were assessed using the eight-item Center for Epidemiological Studies Depression Scale (CES-D8). Housing debt was measured by dummy variables, indicating whether an individual had housing debt, and continuous variables, which were the logarithm of the total amount of housing debt. The two-way fixed effects model was used to examine the relationship. RESULTS: Housing debt had a significant positive impact on depressive symptoms in China. Individuals with housing debt had a 0.176-point higher depressive symptom score than those without housing debt. A 10% increase in the total amount of housing debt led to a 0.16-point increase in depressive symptoms. Non-bank housing loans significantly increased the level of depressive symptoms with a larger coefficient (coef = 0.289), while the impact of bank housing loans was small and not statistically significant. In terms of the types of housing demands, a positive impact was observed only among individuals who had only one property meeting their housing consumption demands. CONCLUSIONS: This study found a significant positive impact of housing debt on depressive symptoms, primarily driven by non-bank housing loans. Furthermore, housing debt increased the depressive symptoms among individuals with consumption demands, while those with investment demands did not show a significant impact. Government interventions should prioritize easing formal financial constraints and providing support for individuals with housing consumption demands.
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Depresión , Vivienda , Humanos , Depresión/epidemiología , Estudios Longitudinales , China/epidemiologíaRESUMEN
The research attention is increasingly directed towards the effective integration of the 17 United Nations Sustainable Development Goals (SDGs) within the limitations of the real world and amidst intersectoral conflicts. In light of the inextricable relationship between irrigation and energy, the objective of this study is to identify potential avenues for achieving the SDG6 and SDG7 goals of enhancing water use efficiency in agriculture and eradicating energy poverty, respectively. Utilizing data from 30 Chinese provinces from 2002 to 2017, this study explores the dynamic influence of energy poverty on agricultural water efficiency with a system generalized method of moments methodology. The findings suggest that energy poverty may greatly reduce agricultural water efficiency. The heterogeneity study shows that when agricultural water efficiency grows, the negative impacts of energy poverty continue to fade. Based on an assessment of various processes, results suggest that non-farm employment and cropping structure modification is a prominent conduit via which energy poverty negatively influences agricultural water efficiency.
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Synchronously improving the photothermal conversion efficiency and photodynamic activity of organic small molecule photosensitizers is crucial for their further wide application in cancer treatment. Recently, the emerging A-D-A photosensitizer-based phototherapy systems have attracted great interest due to their plentiful inherent merits. Herein, we propose a design strategy for A-D-A photosensitizers with synchronously enhanced photothermal conversion and reactive oxygen species (ROS) generation efficiencies. Side chain programming is carried out to design three A-D-A photosensitizers (IDT-H, IDT-Br, IDT-I) containing hexyl, bromohexyl, and iodohexyl side chains, respectively. Theoretical calculations confirm that a bulky iodine atom could weaken the intermolecular π-π stacking and enhance spin-orbit coupling constants of IDT-I. These molecular mechanisms enable IDT-I nanoparticles (NPs) to exhibit 2.4-fold and 1.7-fold higher ROS generation efficiency than that of IDT-H NPs and IDT-Br NPs, respectively, as well as the highest photothermal conversion efficiency. Both the experimental results in vitro and in vivo verify that IDT-I NPs are perfectly qualified for the mission of photothermal and photodynamic synergistic therapy. Therefore, in this contribution, we provide a promising perspective for the design of A-D-A photosensitizers with simultaneously improved photothermal and photodynamic therapy ability.
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An unexpected surge of osteoporosis outpatients occurred after COVID-19 lockdown was lifted in China. To confirm the observation and identify possible reasons driving patients care seeking behaviors post-pandemic, we compared the outpatient volumes at the osteoporosis clinic in January through May, 2019-2022 and surveyed seven osteoporosis specialists across China to validate the phenomenon before devising an online questionnaire to collect patients' characters and physical activity levels. Univariate and binary logistic regression analyses were calculated to identify predictors of post-lockdown care-seeking. We received 480 valid responses, including 397 (82.7%) patients having visited the clinic after lockdown and 83 (17.3%) having not. Four significant predictors were identified, including being female, experiencing pain, aggravating symptoms, and heightened anxiety during lockdown (P < 0.05). Both groups experienced lower physical activity levels during lockdown, which however was not a significant predictor (P = 0.317). The surge in osteoporosis outpatient visits after COVID-19 lockdown suggests vast latent demand for osteoporosis care accumulated during the pandemic. Four significant factors predict post-lockdown outpatient care-seeking, including being female, experiencing pain and aggravating symptoms, and heightened anxiety levels. Though physical activity levels decreased during lockdown, it failed to predict care-seeking. This demonstrates resilience of osteoporosis patients to resume regular care despite disruption and stress the substantial backlog of unmet healthcare needs.
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COVID-19 , Osteoporosis , Humanos , Femenino , Masculino , Pacientes Ambulatorios , Estudios Transversales , COVID-19/epidemiología , Control de Enfermedades Transmisibles , China/epidemiología , Instituciones de Atención Ambulatoria , Osteoporosis/epidemiología , DolorRESUMEN
Background: Retinal detachment (RD) is a common sight-threatening condition in the emergency department. Early postural intervention based on detachment regions can improve visual prognosis. Methods: We developed a weakly supervised model with 24,208 ultra-widefield fundus images to localize and coarsely outline the anatomical RD regions. The customized preoperative postural guidance was generated for patients accordingly. The localization performance was then compared with the baseline model and an ophthalmologist according to the reference standard established by the retina experts. Results: In the 48-partition lesion detection, our proposed model reached an 86.42% (95% confidence interval (CI): 85.81-87.01%) precision and an 83.27% (95%CI: 82.62-83.90%) recall with an average precision (PA) of 0.9132. In contrast, the baseline model achieved a 92.67% (95%CI: 92.11-93.19%) precision and limited recall of 68.07% (95%CI: 67.25-68.88%). Our holistic lesion localization performance was comparable to the ophthalmologist's 89.16% (95%CI: 88.75-89.55%) precision and 83.38% (95%CI: 82.91-83.84%) recall. As to the performance of four-zone anatomical localization, compared with the ground truth, the un-weighted Cohen's κ coefficients were 0.710(95%CI: 0.659-0.761) and 0.753(95%CI: 0.702-0.804) for the weakly-supervised model and the general ophthalmologist, respectively. Conclusion: The proposed weakly-supervised deep learning model showed outstanding performance comparable to that of the general ophthalmologist in localizing and outlining the RD regions. Hopefully, it would greatly facilitate managing RD patients, especially for medical referral and patient education.
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Introduction: Alveolar cleft (AC) is a common congenital defect in people with cleft lip and palate (CLP). Alveolar bone grafting (ABG) is typically performed during adolescence, resulting in the fissure remaining in the mouth for a longer length of time. Patients with AC have a greater rate of oral diseases such as dental caries than the normal population, and the precise characteristics of the bacterial alterations caused by AC are unknown. Methods: We recruited a total of 87 subjects and collected dental plaque samples from AC adolescents (AAP), post-operative ABG adolescents (PAP), healthy control adolescents (CAP), AC young adults (AYP), post-operative ABG young adults (PYP), and healthy control young adults (CYP). The sequencing of 16S rRNA genes was performed. Results: The microbial composition of plaque from alveolar cleft patients differed significantly from age-matched healthy controls. Linear discriminant analysis effect size (LEfSe) analysis revealed that AAP was enriched for Neisseria, Haemophilus, Fusobacterium, Rhodococcus, Aggregatibacter, Gemella, and Porphyromonas, whereas AYP was enriched for Capnocytophaga, Rhodococcus, and Actinomyces-f0332. There were phenotypic differences in facultatively anaerobic, Gram-negative, Gram-positive, and oxidative stress tolerance between the AYP group with longer alveolar cleft and the healthy control group according to Bugbase phenotypic predictions. Alveolar bone grafting did not alter the functional phenotype of alveolar cleft patients but reduced the number of differential genera between alveolar cleft patients and healthy controls at both ages. Conclusions: Our study systematically characterized the supragingival plaque microbiota of alveolar cleft patients, post-alveolar bone grafting patients, and matched healthy controls in two ages to gain a better understanding of plaque ecology and microbiology associated with alveolar clefts.
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Bacterias , Labio Leporino , Fisura del Paladar , Placa Dental , Microbiota , ARN Ribosómico 16S , Humanos , Placa Dental/microbiología , Fisura del Paladar/microbiología , Adolescente , Microbiota/genética , ARN Ribosómico 16S/genética , Femenino , Masculino , Labio Leporino/microbiología , Adulto Joven , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Injerto de Hueso Alveolar , AdultoRESUMEN
BACKGROUND: Transmembrane protein 92 (TMEM92) has been implicated in the facilitation of tumor progression. Nevertheless, comprehensive analyses concerning the prognostic significance of TMEM92, as well as its role in immunological responses across diverse cancer types, remain to be elucidated. METHODS: In this study, data was sourced from a range of publicly accessible online platforms and databases, including TCGA, GTEx, UCSC Xena, CCLE, cBioPortal, HPA, TIMER2.0, GEPIA, CancerSEA, GDSC, exoRBase, and ImmuCellAI. We systematically analyzed the expression patterns of TMEM92 at both mRNA and protein levels across diverse human organs, tissues, extracellular vesicles (EVs), and cell lines associated with multiple cancer types. Subsequently, analyses were conducted to determine the relationship between TMEM92 and various parameters such as prognosis, DNA methylation, copy number variation (CNV), the tumor microenvironment (TME), immune cell infiltration, genes with immunological relevance, tumor mutational burden (TMB), microsatellite instability (MSI), mismatch repair (MMR), and half-maximal inhibitory concentration (IC50) values. RESULTS: In the present study, we observed a pronounced overexpression of TMEM92 across a majority of cancer types, which was concomitantly associated with a less favorable prognosis. A notable association emerged between TMEM92 expression and both DNA methylation and CNV. Furthermore, a pronounced relationship was discerned between TMEM92 expression, the TME, and the degree of immune cell infiltration. Intriguingly, while TMEM92 expression displayed a positive correlation with macrophage presence, it inversely correlated with the infiltration level of CD8 + T cells. Concurrently, significant associations were identified between TMEM92 and the major histocompatibility complex, TMB, MSI, and MMR. Results derived from Gene Set Enrichment Analysis and Gene Set Variation Analysis further substantiated the nexus of TMEM92 with both immune and metabolic pathways within the oncogenic context. CONCLUSIONS: These findings expanded the understanding of the roles of TMEM92 in tumorigenesis and progression and suggest that TMEM92 may have an immunoregulatory role in several malignancies.
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Nonalcoholic fatty liver disease (NAFLD) is the most prevalent type of chronic liver disease. However, the disease is underappreciated as a remarkable chronic disorder as there are rare managing strategies. Several studies have focused on determining NAFLD-caused hepatocyte death to elucidate the disease pathoetiology and suggest functional therapeutic and diagnostic options. Pyroptosis, ferroptosis, and necroptosis are the main subtypes of non-apoptotic regulated cell deaths (RCDs), each of which represents particular characteristics. Considering the complexity of the findings, the present study aimed to review these types of RCDs and their contribution to NAFLD progression, and subsequently discuss in detail the role of necroptosis in the pathoetiology, diagnosis, and treatment of the disease. The study revealed that necroptosis is involved in the occurrence of NAFLD and its progression towards steatohepatitis and cancer, hence it has potential in diagnostic and therapeutic approaches. Nevertheless, further studies are necessary.
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Progresión de la Enfermedad , Necroptosis , Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Ferroptosis , Hepatocitos/patología , Hígado/patología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/diagnóstico , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/terapia , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , PiroptosisRESUMEN
OBJECTIVE: Lennox-Gastaut Syndrome (LGS) is characterized by the presence of multiple seizure types and encompasses a heterogenous group of etiologies. The aim of our study was to evaluate the etiological profile of LGS and investigate seizure outcomes based on different clinical variables. METHODS: The clinical features, neuroimaging findings, genetic testing and other testing results of LGS patients were systematically reviewed. The identifiable etiology was categorized as either acquired or nonacquired. Univariate and multivariate regression analyses were performed to explore the association between clinical variables and seizure outcome at the last follow-up. RESULTS: We enrolled 156 patients diagnosed with LGS, of whom 66% were male. The mean age of patients was 34.2 months and the median follow-up duration was 29.5 months (interquartile range = 14-56.25 months). The initial seizure type was epileptic spasm in 61 patients, among which 33 of them met the criteria for infantile epileptic spasm syndrome. All patients underwent neuroimaging test, with 25% falling into the acquired structural category. Etiology could be identified in 84 individuals, including pathogenetic variants found in 34 out of 117 patients with nonacquired etiology. CHD2 mutations were most frequently observed among these pathogenetic variants. At the last follow-up, favorable outcomes were observed in 27 patients. The identification of etiology emerged as a significant determinant influencing LGS outcome; specifically, patients with unknown etiology had a higher likelihood of experiencing favorable outcomes compared to those with known cause (p = 0.041). Early onset age and longer epilepsy duration significantly increased the odds of an unfavorable outcome (p = 0.006 and 0.024). SIGNIFICANCE: We present novel data on the clinical and etiological spectrum of LGS, with determined etiology observed in over half of the patients. Epileptic spasms were found to be more prevalent than tonic seizures as seizure onset types in LGS. The presence of a known etiology, earlier age at onset, and longer duration of epilepsy were associated with a poorer long-term epileptological outcome.
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Síndrome de Lennox-Gastaut , Humanos , Masculino , Síndrome de Lennox-Gastaut/fisiopatología , Femenino , Preescolar , Lactante , Niño , Estudios de Seguimiento , Estudios Retrospectivos , Espasmos Infantiles/etiología , Espasmos Infantiles/fisiopatología , ElectroencefalografíaRESUMEN
Mango (Mangifera indica L.) has been widely cultivated as a culturally and economically significant fruit tree for roughly 4000 years. Despite its rich history, little is known about the crop's domestication, genomic variation, and the genetic loci underlying agronomic traits. This study employs the whole-genome re-sequencing of 224 mango accessions sourced from 22 countries, with an average sequencing depth of 16.37×, to explore their genomic variation and diversity. Through phylogenomic analysis, M. himalis J.Y. Liang, a species grown in China, was reclassified into the cultivated mango group known as M. indica. Moreover, our investigation of mango population structure and differentiation revealed that Chinese accessions could be divided into two distinct gene pools, indicating the presence of independent genetic diversity ecotypes. By coupling genome-wide association studies with analyses of genotype variation patterns and expression patterns, we identified several candidate loci and dominant genotypes associated with mango flowering capability, fruit weight, and volatile compound production. In conclusion, our study offers valuable insights into the genetic differentiation of mango populations, paving the way for future agronomic improvements through genomic-assisted breeding.
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BACKGROUND: The diagnosis and treatment of attention deficit hyperactivity disorder (ADHD) comorbid with epilepsy have been insufficiently addressed in China. We conducted a study in China to investigate the current status, diagnosis, and treatment of ADHD in children to further our understanding of ADHD comorbid with epilepsy, strengthen its management, and improve patients' quality of life. METHODS: We carried out a multicenter cross-sectional survey of children with epilepsy across China between March 2022 and August 2022. We screened all patients for ADHD and compared various demographic and clinical factors between children with and without ADHD, including gender, age, age at epilepsy onset, duration of epilepsy, seizure types, seizure frequency, presence of epileptiform discharges, and treatment status. Our objective was to explore any possible associations between these characteristics and the prevalence of ADHD. RESULTS: Overall, 395 epilepsy patients aged 6-18 years were enrolled. The age at seizure onset and duration of epilepsy ranged from 0.1-18 to 0.5-15 years, respectively. Focal onset seizures were observed in 212 (53.6%) patients, while 293 (76.3%) patients had epileptiform interictal electroencephalogram (EEG) abnormalities. Among the 370 patients treated with anti-seizure medications, 200 (54.1%) had monotherapy. Although 189 (47.8%) patients had ADHD, only 31 received treatment for it, with the inattentive subtype being the most common. ADHD was more common in children undergoing polytherapy compared to those on monotherapy. Additionally, poor seizure control and the presence of epileptiform interictal EEG abnormalities may be associated with a higher prevalence of ADHD. CONCLUSIONS: While the prevalence of ADHD was higher in children with epilepsy than in normal children, the treatment rate was notably low. This highlights the need to give more importance to the diagnosis and treatment of ADHD in children with epilepsy.
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Radiation-induced intestinal damage (RIID) is a common side effect of radiotherapy in patients with abdominopelvic malignancies. Gap junctions are special structures consisting of connexins (Cxs). This study aimed to investigate the expression and role of connexins in RIID and underlying mechanism. In this study, a calcein-AM fluorescence probe was used to detect changes in gap junctional intercellular communication in intestinal epithelial IEC-6 cells. Our results show that gap junctional intercellular communication of IEC-6 cells was reduced at 6, 12, 24, and 48 h after irradiation, with the most pronounced effect at 24 h. Western blotting and immunofluorescence results showed that the expression of Cx43, but not other connexins, was reduced in irradiated intestinal epithelial cells. Silencing of Cx43 reduced gap junctional intercellular communication between irradiated intestinal epithelial cells with increased ROS and intracellular Ca2+ levels. Furthermore, knockdown of Cx43 reduced the number of clonal clusters, decreased cell proliferation with increased cytotoxicity and apoptosis. Western blotting results showed that silencing of Cx43 resulted in changed γ-H2AX and PI3K/AKT pathway proteins in irradiated intestinal epithelial cells. Administration of the PI3K/AKT pathway inhibitor LY294002 inhibited the radioprotective effects in Cx43-overexpressing intestinal epithelial cells. Our study demonstrated that Cx43 expression is decreased by ionizing radiation, which facilitates the radioprotection of intestinal epithelial cells.
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Conexina 43 , Proteínas Proto-Oncogénicas c-akt , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Calcio/metabolismo , Conexinas/metabolismo , Conexinas/farmacología , Transducción de Señal , Uniones Comunicantes , Comunicación CelularRESUMEN
Background: Tumor mutational burden (TMB) is a valuable prognostic biomarker. This study explored the predictive value of TMB and the potential association between TMB and immune infiltration in diffuse large B-cell lymphoma (DLBCL). Methods: We downloaded the gene expression profile, somatic mutation, and clinical data of DLBCL patients from The Cancer Genome Atlas (TCGA) database. We classified the samples into high-and low-TMB groups to identify differentially expressed genes (DEGs). Functional enrichment analyses were performed to determine the biological functions of the DEGs. We utilized the cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm to estimate the abundance of 22 immune cells, and the significant difference was determined by the Wilcoxon rank-sum test between the high- and low-TMB group. Hub gene had been screened as the prognostic TMB-related immune biomarker by the combination of the Immunology Database and Analysis Portal (ImmPort) database and the univariate Cox analysis from the Gene Expression Omnibus (GEO) database including six DLBCL datasets. Various database applications such as Tumor Immune Estimation Resource (TIMER), CellMiner, konckTF, and Genotype-Tissue Expression (GTEx) verified the functions of the target gene. Wet assay confirmed the target gene expression at RNA and protein levels in DLBCL tissue and cell samples. Results: Single nucleotide polymorphism (SNP) occurred more frequently than insertion and deletion, and C > T was the most common single nucleotide variant (SNV) in DLBCL. Survival analysis showed that the high-TMB group conferred poor survival outcomes. A total of 62 DEGs were obtained, and 13 TMB-related immune genes were identified. Univariate Cox analysis results illustrated that CD1c mutation was associated with lower TMB and manifested a satisfactory clinical prognosis by analysis of large samples from the GEO database. In addition, infiltration levels of immune cells in the high-TMB group were lower. Using the TIMER database, we systematically analyzed that the expression of CD1c was positively correlated with B cells, neutrophils, and dendritic cells and negatively correlated with CD8+ T cells, CD4+ T cells, and macrophages. Drug sensitivity showed a significant positive correlation between CD1c expression level and clinical drug sensitivity from the CellMiner database. CREB1, AHR, and TOX were used to comprehensively explore the regulation of CD1c-related transcription factors and signaling pathways by the KnockTF database. We searched the GETx database to compare the mRNA expression levels of CD1c between DLBCL and normal tissues, and the results suggested a significant difference between them. Moreover, wet experiments were conducted to verify the high expression of CD1c in DLBCL at the RNA and protein levels. Conclusions: Higher TMB correlated with poor survival outcomes and inhibited the immune infiltrates in DLBCL. Our results suggest that CD1c is a TMB-related prognostic biomarker.
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Linfoma de Células B Grandes Difuso , Humanos , Algoritmos , Linfocitos B , Biomarcadores , Linfoma de Células B Grandes Difuso/genética , ARNRESUMEN
Trichogramma, an effective biological control agent, demonstrates promise in environmentally sustainable pest management through its parasitic action toward insect eggs. This study evaluates the parasitism fitness and ability of T. chilonis with regard to two factitious host eggs, aiming to develop a cost-effective biological control program. While T. chilonis demonstrated the ability to parasitize both host eggs, the results indicate a preference for ES eggs over COS eggs. The parasitism and emergence rates of T. chilonis regarding ES eggs (parasitism: 89.3%; emergence: 82.6%) surpassed those for COS eggs (parasitism: 74.7%; emergence: 68.8%), with a notable increase in the number of emergence holes observed in the ES eggs compared to the COS eggs. Moreover, the developmental time of T. chilonis for ES eggs (10.8 days) was shorter than that for COS eggs (12.5 days), resulting in a lower number of dead wasps produced. Notably, no significant difference was observed in the female ratios between the two species. A comprehensive analysis was conducted, comparing the size and shell thickness of the two factitious hosts. The ES eggs exhibited smaller dimensions (length: 1721.5 µm; width: 1178.9 µm) in comparison to the COS eggs (length: 2908.8 µm; width: 2574.4 µm), with the ES eggshells being thinner (33.8 µm) compared to the COS eggshells (47.3 µm). The different host species had an effect on the body length of the reared parasitoids, with T. chilonis reared on COS hosts exhibiting a larger body length (female: 626.9 µm; male: 556.7 µm) than those reared on ES hosts (female: 578.8 µm; male: 438.4 µm). Conclusively, the results indicate that ES eggs present a viable alternative to COS eggs for the mass production of Trichogramma species in biological control programs.