Biology subjects

Sun, C.

Publications and source records attributed to Sun, C..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

3D Printing of Multiscale Scaffolds with Microtopography for Guiding Tissue Organization and Regeneration

Engineering biomaterial scaffolds with multiscale structures, integrating anatomically accurate macroscale architecture (millimeters to centimeters) with microtopographic features (sub-microns to tens of microns), is critical for guiding cellular organization and tissue regeneration. However, fabricating such multiscale scaffolds remains challenging due to the limitations of conventional manufacturing techniques and the trade-off between speed and resolution in current 3D printing methods. Here, we present a multiscale micro-continuous liquid interface production (M{micro}CLIP) technique that enables rapid, one-step 3D printing of centimeter-scale scaffolds with spatially tunable microtopography of various sizes and geometries in just a few minutes (up to 1 mm/min). To showcase the versatility of our technique, we printed a one-centimeter-long tubular scaffold with dual microtopographic patterns, i.e. 20 {micro}m axially aligned grooves on the internal surface and 15 {micro}m circumferentially aligned rings on the external surface. These scaffolds induced the simultaneous orientation of vascular endothelial cells along the axial grooves and vascular smooth muscle cells along the circumferential rings, mimicking the orthogonally aligned bilayer architecture of natural arteries. Moreover, the groove patterns significantly accelerated endothelial cell migration, potentially enhancing endothelialization in vascular implants. This approach provides a versatile tool for designing advanced scaffolds and medical devices that harness microtopography to guide tissue organization and enhance regeneration.

bioengineering↗

bioRxiv2024

Robotic Visible-Light Optical Coherence Tomography Visualizes Segmental Schlemms Canal Anatomy and Segmental Pilocarpine Response

PurposeTo use robotic visible-light OCT (vis-OCT) to study circumferential segmental Schlemms canal (SC) anatomy in mice after topical pilocarpine administration. MethodsAnterior segment imaging was performed using a vis-OCT sample arm attached to a 6-degree-of-freedom robotic arm to maintain normal (perpendicular) laser illumination aimed at SC around the limbus. Sixteen mice were studied for repeatability testing and to study aqueous humor outflow (AHO) pathway response to topical drug. Pharmaceutical-grade pilocarpine (1%; n = 5) or control artificial tears (n = 9) were given, and vis-OCT imaging was performed before and 15 minutes after drug application. After SC segmentation, SC areas and volumes were measured circumferentially in control- and drug-treated eyes. ResultsCircumferential vis-OCT provided high-resolution imaging of the anterior segment and AHO pathways, including SC. Segmental SC anatomy was visualized with the average cross-sectional area greatest temporal (3971 {+/-} 328 {micro}m2) and the least nasal (2727 {+/-} 218 {micro}m2; p = 0.018). After pilocarpine administration, the iris became flatter, and SC became larger (pilocarpine: 26.8 {+/-} 5.0% vs. control: 8.9 {+/-} 4.6% volume increase; p = 0.030). However, the pilocarpine alteration was segmental as well, with a greater increase observed superior (pilocarpine: 31.6 {+/-} 8.9% vs. control: 1.8 {+/-} 5.7% volume increase; p = 0.023) and nasal (pilocarpine: 41.1 {+/-} 15.3% vs. control: 13.9 {+/-} 4.5% volume increase; p = 0.045). ConclusionHigh-resolution circumferential non-invasive imaging using AS-OCT of AHO pathways is possible in living animals with robotic control. Segmental SC anatomy was seen at baseline and was consistent with the known segmental nature of trabecular AHO. Segmental SC anatomical response to a muscarinic agonist was seen as well. Segmental glaucoma drug response around the circumference of AHO pathways is a novel observation that may explain the variable patient response to glaucoma treatments.

bioengineering↗

bioRxiv2024

Deep Generative Optimization of mRNA Codon Sequences for Enhanced Protein Production and Therapeutic Efficacy

Messenger RNA (mRNA) therapeutics show immense promise, but their efficacy is limited by suboptimal protein expression. Here, we present RiboDecode, a deep learning framework that generates mRNA codon sequences for enhanced mRNA translation. RiboDecode introduces several advances, including direct learning from large-scale ribosome profiling data and generative exploration of a large sequence space. In silico analysis demonstrates RiboDecode s robust predictive accuracy for unseen genes and cellular environments. In vitro experiments showed substantial improvements in protein expression, significantly outperforming past methods. In addition, RiboDecode enables mRNA design with consideration of cellular context and demonstrates robust performance across different mRNA formats, including m1{Psi}-modified and circular mRNAs, an important feature for mRNA therapeutics. In vivo mouse studies showed that optimized influenza hemagglutinin mRNAs induce ten times stronger neutralizing antibody responses against influenza virus compared to the unoptimized sequence. In an optic nerve crush model, optimized nerve growth factor mRNAs achieve equivalent neuroprotection of retinal ganglion cells at one-fifth the dose of the unoptimized sequence. Collectively, RiboDecode represents a paradigm shift from rule-based to a data-driven, context-aware approach for mRNA therapeutic applications, enabling the development of more potent and dose-efficient treatments.

bioinformatics↗

bioRxiv2024

Decreased Astrocytic CCL5 by MiR-324-5p Ameliorates Ischemic Stroke Injury via CCR5/ERK/CREB Pathway

Following ischemic stroke, Ccl5 mRNA expression increased, while miR-324-5p expression decreased in the peri-infarct cortex of middle cerebral artery occlusion (MCAO) mice. However, the roles of CCL5 and miR-324-5p in stroke remained largely unclear. Here, we show that inhibiting CCL5 using antibodies or miR-324-5p not only reduced infarct area and preserved neurological function in MCAO mice but also attenuated astrocyte reactivity and microglial activation, protected dendritic structures, and maintained spine density. In an astrocyte-neuron co-culture system after oxygen-glucose deprivation (OGD), inhibiting astrocytic CCL5 by antibody or miR-324-5p decreased neuronal apoptosis and preserved dendritic structure. Importantly, the suppression of CCL5 enhanced ERK/CREB pathway signaling both in vivo and in vitro. Consistent with these findings, the application of Maraviroc, a CCR5 antagonist, reduced infarct size, decreased neuronal apoptosis, and upregulated the ERK/CREB pathway in neurons treated with OGD. In conclusion, targeting the CCL5 pathway via miR-324-5p represents a promising therapeutic strategy for alleviating ischemic stroke damage through modulation of the neuronal CCR5/ERK/CREB pathway.

neuroscience↗

bioRxiv2024

Genetic differentiation and adaptive evolution of buff-tailed bumblebees in Asia

Bumblebees are ecologically and economically important pollinating insects, so their declines resulting from environmental changes have received intensive attention. Understanding how environmental factors shape the genetic differentiation of natural populations and identifying the genetic basis of local adaptation will provide insights into how species may cope with environmental changes. The buff-tailed bumblebee (Bombus terrestris) has a wide natural distribution range and has been successfully domesticated to produce commercial colonies for greenhouse pollination. Previous population genetics studies on B. terrestris have mainly focused on populations in Europe; however, populations in Asia, representing the eastern side of its natural distribution, have been less thoroughly sampled. To fill this gap, we collected wild B. terrestris samples from Asia, as well as wild B. terrestris from Europe and samples from domesticated colonies. We conducted whole-genome resequencing for 77 collected B. terrestris workers and performed population genomics analysis. Our results revealed significant genetic differentiation (FST= 0.076) between buff-tailed bumblebees in Europe and Asia, along with notable morphological and physiological differences. Consequently, B. terrestris in Asia represents a distinct new genetic resource. Demographic analysis suggested that the population size of buff-tailed bumblebees had increased during historic cold periods, confirming their cold-adapted characteristics. Selective sweep analysis identified 331 genes under selection in the genomes of Asian B. terrestris, likely involved in their adaptation to high ultraviolet radiation, low temperature, and low precipitation in their habitats. Our research provides insights into the population genetic structure and genetic basis of local adaptation in the buff-tailed bumblebee, which will be useful for its conservation and management.

bioinformatics↗

bioRxiv2024

Implementation and calibration of spectroscopic single-molecule localization microscopy

Spectroscopic single-molecule localization microscopy (sSMLM) allows multi-color super-resolution images with high spectral sensitivity. In this protocol, we provide essential information for researchers to implement sSMLM in a laboratory setting. We describe how to assemble and align the illumination and detection paths of a 3D dual-wedge prism (DWP)-based sSMLM instrument. We provide detailed step-by-step instructions for performing spectral and axial calibration using fluorescent beads and a nanohole array, respectively. We also discuss using sSMLM to image fluorescently labeled cells and report a new MATLAB package, RainbowSTORM v2, to reconstruct super-resolution 3D images. Further, we present representative images as typical anticipated results for users to validate.

bioengineering↗

bioRxiv2024

Genome-based discovery of pachysiphine synthases in Tabernaemontana elegans

Plant specialized metabolism represents an inexhaustible source of active molecules, some of which have been used in human health for decades. Among these, monoterpene indole alkaloids (MIAs) include a wide range of valuable compounds with anticancer, antihypertensive, or neuroactive properties. This is particularly the case for the pachysiphine derivatives which show interesting antitumor and anti-alzheimer activities but accumulate at very low levels in several Tabernaemontana species. Unfortunately, genome data in Tabernaemontanaceae are lacking and knowledge on the biogenesis of pachysiphine-related MIAs in planta remains scarce, limiting the prospects for biotechnological supply of many pachysiphine-derived biopharmaceuticals. Here, we report a raw version of the toad tree (Tabernaemontana elegans) genome sequence. These new genomic resources led to the identification and characterization of a couple of genes encoding cytochrome P450 with pachysiphine synthase activity. Our phylogenomic and docking analyses highlights the different evolutionary processes that have been recruited to epoxidize the pachysiphine precursor tabersonine at a specific position and in a dedicated orientation, thus enriching our understanding of the diversification and speciation of the MIA metabolism in plants. These gene discoveries also allowed us to engineer the synthesis of MIAs in yeast through the combinatorial association of metabolic enzymes resulting in the tailor-made synthesis of non-natural MIAs. Overall, this work represents a step forward for the future supply of pachysiphine-derived drugs by microbial cell factories. Significance StatementWhile pachysiphine is a monoterpene indole alkaloid of high interest and the precursor of an anti-Alzheimer compound, its biosynthesis involving the epoxidation of tabersonine remains uncharacterized. By sequencing and assembling the genome of Tabernaemontana elegans, we identified two P450s exhibiting a pachysiphine synthase activity that we modelized to explore the evolutionary scenario leading to the acquisition of this expoxidase activity; and used to engineer yeast cell factories for securing pachysiphine supply and producing new-to-nature alkaloids.

plant biology↗

bioRxiv2024

In vivo quantification of anterior and posterior chamber volumes in mice: implications for aqueous humor dynamics

PurposeAqueous humor inflow rate, a key parameter influencing aqueous humor dynamics, is typically measured by fluorophotometery. Analyzing fluorophotometric data depends, inter alia, on the volume of aqueous humor in the anterior, but not the posterior, chamber. Previous fluorophotometric studies of aqueous inflow rate in mice have assumed the ratio of anterior:posterior volumes in mice to be similar to those in humans. Our goal was to measure anterior and posterior chamber volumes in mice to facilitate better estimates of aqueous inflow rates. MethodsWe used standard near-infrared optical coherence tomography (OCT) and robotic visible-light OCT (vis-OCT) to visualize, reconstruct and quantify the volumes of the anterior and posterior chambers of the mouse eye in vivo. We used histology and micro-CT scans to validate relevant landmarks from ex vivo tissues to facilitate in vivo measurement. ResultsPosterior chamber volume is 1.1 times the anterior chamber volume in BALB/cAnNCrl mice, i.e. the anterior chamber constitutes about 47% of the total aqueous humor volume, which is very dissimilar to the situation in humans. Anterior chamber volumes in 2-month-old BALB/cAnNCrl and 7-month-old C57BL6/J mice were 1.55 {+/-} 0.36 {micro}L (n=10) and 2.41 {+/-} 0.29 {micro}L (n=8), respectively. This implies that previous studies likely over-estimated aqueous inflow rate by approximately two-fold. ConclusionsIt is necessary to reassess previously reported estimates of aqueous inflow rates, and thus aqueous humor dynamics in the mouse. For example, we now estimate that only 0-15% of aqueous humor drains via the pressure-independent (unconventional) route, similar to that seen in humans and monkeys.

physiology↗

bioRxiv2024

Size evolution of gigantic genomes suggests stochastic outcomes of transposable element/host silencing interactions

Size evolution among gigantic genomes involves gain and loss of many gigabases of transposable elements (TEs), sequences that parasitize host genomes. Animals suppress TEs using piRNA and KRAB-ZFP pathways. TEs and hosts coevolve in an arms race, where suppression strength reflects TE fitness costs. In enormous genomes, additional TE costs become miniscule. How, then, do TEs and host suppression invoke further addition of massive DNA amounts? We analyzed TE proliferation histories, deletion rates, and community diversities in six salamander genomes (21.3 - 49.9 Gb), alongside gonadal expression of TEs and suppression pathways. TE activity is higher in testes than ovaries, attributable to lower KRAB-ZFP suppression. Unexpectedly, genome size/expansion is uncorrelated with TE deletion rate, proliferation history, expression, and host suppression. Also, TE community diversity increases with genome size, contrasting theoretical predictions. TE/host antagonism in gigantic genomes likely produces stochastic TE accumulation, determined by noisy intermolecular interactions in huge genomes/cells.

evolutionary biology↗

bioRxiv2024

APP beta-CTF triggers cell-autonomous synaptic toxicity independent of Abeta

A{beta} is believed to play a significant role in synaptic degeneration observed in Alzheimers disease (AD) and is primarily investigated as a secreted peptide. However, the contribution of intracellular A{beta} or other cleavage products of its precursor protein (APP) to synaptic loss remains uncertain. In this study, we conducted a systematic examination of their cell-autonomous impact using a sparse expression system. Here, these proteins/peptides were overexpressed in a single neuron, surrounded by thousands of untransfected neurons. Surprisingly, we found that APP induced dendritic spine loss only when co-expressed with BACE1. This effect was mediated by {beta}-CTF, a {beta}-cleavage product of APP, through an endosome-related pathway independent of A{beta}. Neuronal expression of {beta}-CTF in mouse brains resulted in defective synaptic transmission and cognitive impairments, even in the absence of amyloid plaques. These findings unveil a {beta}-CTF-initiated mechanism driving synaptic toxicity irrespective of amyloid plaque formation and suggest a potential intervention by inhibiting the endosomal GTPase Rab5.

neuroscience↗

bioRxiv2024

STHD: probabilistic cell typing of single Spots in whole Transcriptome spatial data with High Definition

Recent spatial transcriptomics (ST) technologies have enabled single- and sub-cellular resolution profiling of gene expression across the whole transcriptome. However, the transition to high-definition ST significantly increased data sparsity and dimensionality, posing computational challenges in identifying cell types, deciphering neighborhood structure, and detecting differential expression - all are crucial steps to study normal and disease ST samples. Here we present STHD, a novel machine learning method for probabilistic cell typing of single spots in whole-transcriptome, high-resolution ST data. Unlike the current binning-aggregation-deconvolution strategy, STHD directly models gene expression at single-spot level to infer cell type identities without cell segmentation or spot aggregation. STHD addresses sparsity by modeling count statistics, incorporating neighbor similarities, and leveraging reference single-cell RNA-seq data. We show in VisiumHD data that STHD accurately predicts cell type identities at single-spot level, which achieves precise segmentation of both global tissue architecture and local multicellular neighborhoods. The high-resolution labels facilitate various downstream analyses, including cell type-stratified bin aggregation, spatial compositional comparisons, and cell type-specific differential expression analyses. Moreover, STHD labels further reveal frontlines of inter-cell type interactions at immune hubs in cancer samples. STHD is scalable and generalizable across diverse samples, tissues, and diseases, facilitating genome-wide analyses in various spatial organization contexts. Overall, computational modeling of individual spots with STHD facilitates discoveries in cellular interactions and molecular mechanisms in whole-genome spatial technologies with high resolution. STHD is available at https://github.com/yi-zhang/STHD.

bioinformatics↗

bioRxiv2024

GCRTcall: a Transformer based basecaller for nanopore RNA sequencing enhanced by gated convolution and relative position embedding via joint loss training

Nanopore sequencing, renowned for its ability to sequence DNA and RNA directly with read lengths extending to several hundred kilobases or even megabases, holds significant promise in fields like transcriptomics and other omics studies. Despite its potential, the technologys limited accuracy in base identification has restricted its widespread application. Although many algorithms have been developed to improve DNA decoding, advancements in RNA sequencing remain limited. Addressing this challenge, we introduce GCRTcall, a novel approach integrating Transformer architecture with gated convolutional networks and relative positional encoding for RNA sequencing signal decoding. Our evaluation demonstrates that GCRTcall achieves state-of-the-art performance in RNA basecalling.

bioinformatics↗

bioRxiv2024

BaseNet: A Transformer-Based Toolkit for Nanopore Sequencing Signal Decoding

Nanopore sequencing provides a rapid, convenient and high-throughput solution for nucleic acid sequencing. Accurate basecalling in nanopore sequencing is crucial for downstream analysis. Traditional approaches such as Hidden Markov Models (HMM), Recurrent Neural Networks (RNN), and Convolutional Neural Networks (CNN) have improved basecalling accuracy but there is a continuous need for higher accuracy and reliability. In this study, we introduce BaseNet, an open-source toolkit that utilizes transformer models for advanced signal decoding in nanopore sequencing. BaseNet incorporates both autoregressive and non-autoregressive transformer-based decoding mechanisms, offering state-of-the-art algorithms freely accessible for future improvement. Our research indicates that cross-attention weights effectively map the relationship between current signals and base sequences, joint loss training through adding a pair of forward and reverse decoder facilitate model converge, and large-scale pre-trained models achieve superior decoding accuracy. This study helps to advance the field of nanopore sequencing signal decoding, contributes to technological advancements, and provides novel concepts and tools for researchers and practitioners.

bioinformatics↗

bioRxiv2024

Development of non-β-Lactam covalent allosteric inhibitors targeting PBP2a in Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive bacterial pathogen, continues to pose a serious threat to the current public health system in our society. The high level of resistance to {beta}-lactam antibiotics in MRSA is attributed to the expression of penicillin-binding protein 2a (PBP2a), which catalyzes cell wall cross-linking. According to numerous research reports, the activity of the PBP2a protein is known to be regulated by an allosteric site distinct from the active site where cell wall cross-linking occurs. Here, we conducted a screening of 113 compounds containing a 1,3,4-oxadiazole core to design new covalent inhibitors targeting the allosteric site of PBP2a and establish their structural-activity relationship. The stereochemically selective synthesis of sulfonyl oxadiazole compounds identified in the initial screening resulted in a maximum eightfold enhancement in cell inhibition activity. The sulfonyl oxadiazole-based compounds formulated as PEG-based ointments, with low toxicity test results on human cells (CC50: >78M), demonstrated potent antimicrobial effects not only in a mouse skin wound infection model but also against oxacillin-resistant clinical isolate MRSA (IC50 {approx} 1M), as evidenced by the results. Furthermore, additional studies utilizing LC-MS/MS and in-silico approaches clearly support the allosteric site covalent binding mechanism through the nucleophilic aromatic substitution (SNAr) reaction, as well as its association with the closure of the major active site of PBP2a.

microbiology↗

bioRxiv2024

Maximizing photon utilization in spectroscopic single-molecule localization microscopy using symmetrically dispersed dual-wedge prisms

Single-molecule localization microscopy (SMLM) enables super-resolution imaging on conventional fluorescent microscopes. Spectroscopic SMLM (sSMLM) further allows highly multiplexed super-resolution imaging. We report an easy-to-implement symmetrically dispersed dual-wedge prism (SDDWP)-sSMLM design that maximizes photon utilization. We first symmetrically dispersed photons to the -1st and +1st orders in an optical assembly using two identical dual-wedge prisms (DWPs). Then we computationally extracted the fluorophores spatial position and spectral characteristics using photons in both the -1st and +1st orders. Theoretical analysis and experimental validation showed lateral and spectral precisions of 10.1 nm and 0.3 nm, respectively, representing improvements of 28% and 48% over our previous DWP-based system, where emitted photons are divided separately for spatial and spectral analyses.

bioengineering↗

bioRxiv2024

A Novel Liver Cancer-Selective Histone Deacetylase Inhibitor Is Effective Against Hepatocellular Carcinoma and Induces Durable Responses with Immunotherapy

Hepatocellular cancer (HCC) progression is facilitated by gene-silencing chromatin histone hypoacetylation due to histone deacetylases (HDACs) activation. However, inhibiting HDACs -- an effective treatment for lymphomas -- has shown limited success in solid tumors. We report the discovery of a class of HDAC inhibitors (HDACi) that demonstrates exquisite selective cytotoxicity against human HCC cells. The lead compound STR-V-53 (3) showed a favorable safety profile in mice and robustly suppressed tumor growth in orthotopic xenograft models of HCC. When combined with the anti-HCC drug sorafenib, STR-V-53 showed greater in vivo efficacy. Moreover, STR-V-53 combined with anti-PD1 therapy increased the CD8+ to regulatory T-cell (Treg) ratio and survival in an orthotopic HCC model in immunocompetent mice. This combination therapy resulted in durable responses in 40% of the mice. Transcriptomic analysis revealed that STR-V-53 primed HCC cells to immunotherapy through HDAC inhibition, impaired glucose-regulated transcription, impaired DNA synthesis, upregulated apoptosis, and stimulated the immune response pathway. Collectively, our data demonstrate that the novel HDACi STR-V-53 is an effective anti-HCC agent that can induce profound responses when combined with standard immunotherapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/587062v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@28281corg.highwire.dtl.DTLVardef@617aa9org.highwire.dtl.DTLVardef@1bc198dorg.highwire.dtl.DTLVardef@31852e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

TAK1 is a serine/threonine protein kinase that is a key regulator in a wide variety of cellular processes. However, the functions and mechanisms involved in cancer metastasis are still not well understood. Here, we found that TAK1 knockdown promoted esophageal squamous cancer cell (ESCC) migration and invasion, whereas TAK1 overexpression resulted in the opposite outcome. These in vitro findings were recapitulated in vivo in a xenograft metastatic mouse model. Mechanistically, co-immunoprecipitation and mass spectrometry demonstrated that TAK1 interacted with phospholipase C epsilon 1 (PLCE1), and phosphorylated PLCE1 at serine 1060 (S1060). Functional studies revealed that phosphorylation at S1060 in PLCE1 resulted in decreased enzyme activity, leading to the repression of PIP2 hydrolysis. As a result, the degradation products of PIP2 including diacylglycerol (DAG) and inositol IP3 were reduced, which thereby suppressed signal transduction in the axis of PKC/GSK-3{beta}/{beta}-Catenin. Consequently, expression of cancer metastasis-related genes was impeded by TAK1. Overall, our data indicate that TAK1 plays a negative role in ESCC metastasis, which depends on the TAK1 induced phosphorylation of PLCE1 at S1060.

cancer biology↗

bioRxiv2024

Phylogenomics resolves a 100-year-old debate regarding the evolutionary history of caddisflies (Insecta: Trichoptera)

Trichoptera (caddisfly) phylogeny provides an interesting example of aquatic insect evolution, with rich ecological diversification, especially for underwater architecture. Trichoptera provide numerous critical ecosystem services and are also one of the most important groups of aquatic insects for assessing water quality. The phylogenetic relationships of Trichoptera have been debated for nearly a century. In particular, the phylogenetic position of the "cocoon-makers" within Trichoptera has long been contested. Here, we designed a universal single-copy orthologue and sets of ultraconserved element markers specific for Trichoptera for the first time. Simultaneously, we reconstructed the phylogenetic relationship of Trichoptera based on genome data from 111 species, representing 29 families and 71 genera. Our phylogenetic inference clarifies the probable phylogenetic relationships of "cocoon-makers" within Integripalpia. Hydroptilidae is considered as the basal lineage within Integripalpia, and the families Glossosomatidae, Hydrobiosidae, and Rhyacophilidae formed a monophyletic clade, sister to the integripalpian subterorder Phryganides. The resulting divergence time and ancestral state reconstruction suggest that the most recent common ancestor of Trichoptera appeared in the early Permian and that diversification was strongly correlated with habitat change.

zoology↗