The involvement of VEGF and VEGFR in bacterial recognition and regulation of antimicrobial peptides in Eriocheir sinensis.

Vascular endothelial growth factor (VEGF) and VEGF reporter (VEGFR) are essential molecules in VEGF signalling pathway. Although the functions of VEGF and VEGFR have been well reported in vertebrates, their functions are still poorly understood in invertebrates. In this study, the open reading frame sequences of EsVEGF1 and EsVEGFR4 were cloned from Eriocheir sinensis, and their corresponding proteins shared typical structure characteristics with their counterparts in other species. EsVEGF1 were predominantly expressed in hepatopancreas and muscle while EsVEGFR4 mainly expressed in hemocytes and intestine. The expression levels of EsVEGF1 in hemocytes were rapidly induced by Staphylococcus aureus and Vibrio parahaemolyticus, and it also increased rapidly in hepatopancreas after being challenged with V. parahaemolyticus. The expression levels of EsVEGFR4 only increased in hepatopancreas of crabs injected with S. aureus. The extracellular immunoglobulin domain of EsVEGFR4 could bind with Gram-negative and Gram-positive bacteria as well as lipopolysaccharide and peptidoglycan. EsVEGF1 could act as the ligand for EsVEGFR4 and Toll-like receptor and regulate the expression of crustins and lysozyme with a tissue-specific manner, while have no regulatory function on that of anti-lipopolysaccharide factors. This study will provide new insights into the immune defense mechanisms mediated by VEGF and VEGFR in crustaceans.

Analysis of adaptive molecular mechanisms in response to low salinity in antennal gland of mud crab, Scylla paramamosain.

As an important marine aquaculture species, the mud crab (Scylla paramamosain) is a good candidate for studying the osmoregulatory mechanism of crustaceans. While previous studies have focused on the osmoregulatory function of the gills, this study aims to explore the osmoregulatory function of the antennal glands. By the comparative transcriptomic analysis, we found the pathways of ion regulation including "proximal tubule bicarbonate reclamation" and "mineral absorption" were activated in the antennal glands of the crabs long-term dwelling in low salinity. The enhanced ionic reabsorption was associated with up-regulated ion transport genes such as NKA, CA-c, VPA, and NHE, and with energy metabolism genes such as MDH, SLC25, and PEPCK. The upregulation of NKA and CA-c was also verified by the increased enzyme activity. The lowered osmolality and ion concentration of the hemolymph and the enlarged labyrinth lumen and hemolymph capillary inside the antennal glands indicated the infiltration of external water and the responsively increase of urine excretion, which explained the requirement of enhanced ionic reabsorption. To further confirm these findings, we examined the change of gene expression, enzyme activity, internal ion concentration, and external ion concentration during a 96 h low salinity challenge with seven intervals. The results were basically consistent with the results as shown in the long-term low salinity adaptation. The present study provides valuable information on the osmoregulatory function of the antennal glands of S. paramamosain. The implication of this study in marine aquaculture is that it provides valuable information on the osmoregulatory mechanism of mud crabs, which can be used to improve their culture conditions and enhance their tolerance to salinity stress. The identified genes and pathways involved in osmoregulation can also be potential targets for genetic selection and breeding programs to develop more resilient mud crab strains for aquaculture.

Identifying low salinity adaptation gene expression in the anterior and posterior gills of the mud crab (Scylla paramamosain) by transcriptomic analysis.

In the present study, BGISEQ-500 RNA-Seq technology was adopted to investigate how Scylla paramamosain adapts to salinity tolerance at the molecular level and explores changes in gene expression linked to salinity adaptation following exposure to both low salinity (5 ‰) and standard salinity (23 ‰) conditions. A total of 1100 and 520 differentially expressed genes (DEGs) were identified in the anterior and posterior gills, respectively, and their corresponding expression patterns were visualized in volcano plots and a heatmap. Further analysis highlighted significant enrichment of well-established gene functional categories and signaling pathways, including those what associated with cellular stress response, ion transport, energy metabolism, amino acid metabolism, H2O transport, and physiological stress compensation. We also selected key DEGs within the anterior and posterior gills that encode pivotal stress adaptation and tolerance modulators, including AQP, ABCA1, HSP 10, A35, CAg, NKA, VPA, CAc, and SPS. Interestingly, A35 in the gills might regulate osmolality by binding CHH in response to low salinity stress or serve as a mechanism for energy compensation. Taken together, our findings elucidated the intricate molecular mechanism employed by S. paramamosain for salinity adaptation, which involved distinct gene expression patterns in the anterior and posterior gills. These findings provide the foothold for subsequent investigations into salinity-responsive candidate genes and contribute to a deeper understanding of S. paramamosain's adaptation mechanisms in low-salinity surroundings, which is crucial for the development of low-salinity species cultivation and the establishment of a robust culture model.

Activation and characterization of G protein-coupled receptors for CHHs in the mud crab, Scylla paramamosain.

Crustacean hyperglycemic hormone (CHH) superfamily peptides constitute a group of neurohormones, including the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), which reportedly play an essential role in regulating various biological activities by binding to their receptors in crustaceans. Although bioinformatics analyses have identified G protein-coupled receptors (GPCRs) as potential CHH receptors, no validation through binding experiments has been carried out. This study employed a eukaryotic expression system, HEK293T cell transient transfection, and ligand-receptor interaction tests to identify the GPCRs of CHHs in the mud crab Scylla paramamosain. We found that four GPCRs (Sp-GPCR-A34-A37) were activated by their corresponding CHHs (Sp-CHH1-v1, Sp-MIH, Sp-VIH) in a dose-dependent manner. Of these, Sp-GPCR-A34 was exclusively activated by Sp-VIH; Sp-GPCR-A35 was activated by Sp-CHH1-v1 and Sp-VIH, respectively; Sp-GPCR-A36 was activated by Sp-CHH1-v1 and Sp-MIH; Sp-GPCR-A37 was exclusively activated by Sp-MIH. The half-maximal effective concentration (EC50) values for all CHHs/GPCRs pairs (both Ca2+ and cAMP signaling) were in the nanomolar range. Overall, our study provided hitherto undocumented evidence of the presence of G protein-coupled receptors of CHH in crustaceans, providing the foothold for further studies on the signaling pathways of CHHs and their corresponding GPCRs.

Expression characteristics and inhibitory activity of a leucine-rich repeat (LRR)-only protein in the Chinese mitten crab, Eriocheir sinensis.

The leucine-rich repeat (LRR) domain is a crucial structure in a variety of immune related proteins and displays multiple immune functions. In this study, the open reading frame (ORF) of an LRR-only protein was cloned from the Chinese mitten crab, Eriocheir sinensis (EsLRRop1). The protein sequence of EsLRRop1 contained seven LRR motifs, three LRR-TYP motifs and an LRRCT motif. Tissue distribution exhibited that EsLRRop1 mainly expressed in nervous tissues including thoracic ganglion, eyestalk and brain while showed relatively lower transcriptional level in hemocyte. Based on the above expression characteristics, the responses of EsLRRop1 to the challenge of Vibrio parahaemolyticus and Staphylococcus aureus were tested. The result showed that the transcript of EsLRRop1 in thoracic ganglion and eyestalk up-regulated after being challenged with S. aureus, while it decreased post injection with V. parahaemolyticus. The transcript of EsLRRop1 in hemocytes up-regulated sharply at 3 h and decreased at 12 h and 24 h after being challenged with V. parahaemolyticus, while it decreased at 12 h and 24 h post injection with S. aureus. The recombinant protein of EsLRRop1 (His-EsLRRop1) displayed binding activities to V. alginolyticus, V. harveyi, V. parahaemolyticus, S. aureus, Corynebacterium glutamicum and Micrococcus lysodeikticus as well as lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, the His-EsLRRop1 exhibited inhibitory activity against V. parahaemolyticus and V. harveyi with minimum inhibitory concentration (MIC) of 3.57-7.14 µM and 7.14-14.28 µM, respectively. These results provide theoretical basis for the application of EsLRRop1 in inhibiting bacteria in aquaculture practice.

Comparative Analysis of Transposable Elements Reveals the Diversity of Transposable Elements in Decapoda and Their Effects on Genomic Evolution.

Transposable elements (TEs) are mobile genetic elements that exist in the host genome and exert considerable influence on the evolution of the host genome. Since crustaceans, including decapoda, are considered ideal models for studying the relationship between adaptive evolution and TEs, TEs were identified and classified in the genomes of eight decapoda species and one diplostraca species (as the outgroup) using two strategies, namely homology-based annotation and de novo annotation. The statistics and classification of TEs showed that their proportion in the genome and their taxonomic composition in decapoda were different. Moreover, correlation analysis and transcriptome data demonstrated that there were more PIF-Harbinger TEs in the genomes of Eriocheir sinensis and Scylla paramamosain, and the expression patterns of PIF-Harbingers were significantly altered under air exposure stress conditions. These results signaled that PIF-Harbingers expanded in the genome of E. sinensis and S. paramamosain and might be related to their air exposure tolerance levels. Meanwhile, sequence alignment revealed that some Jockey-like sequences (JLSs) with high similarity to specific regions of the White spot syndrome virus (WSSV) genome existed in all eight decapod species. At the same time, phylogenetic comparison exposed that the phylogenetic tree constructed by JLSs was not in agreement with that of the species tree, and the distribution of each branch was significantly different. The abovementioned results signaled that these WSSV-specific JLSs might transfer horizontally and contribute to the emergence of WSSV. This study accumulated data for expanding research on TEs in decapod species and also provided new insights and future direction for the breeding of stress-resistant and disease-resistant crab breeds.

Eriocheir sinensis feminization-1c (Fem-1c) and Its Predicted miRNAs Involved in Sexual Development and Regulation.

Feminization-1c (Fem-1c) is important for sex differentiation in the model organism Caenorhabditis elegans. In our previous study, the basic molecular characteristics of the Fem-1c gene (EsFem-1c) in Eriocheir sinensis (Henri Milne Edwards, 1854) were cloned to determine the relationship with sex differentiation. In this study, the genomic sequence of EsFem-1c contained five exons and four introns, with an exceptionally long 3'UTR sequence. The qRT-PCR results of EsFem-1c demonstrated lower tissue expression in the androgenic gland of the intersex crab than the normal male crab, implying that EsFem-1c plays a role in crab AG development. RNA interference experiments and morphological observations of juvenile and mature crabs indicated that EsFem-1c influences sexual development in E. sinensis. A dual-luciferase reporter assay disclosed that tcf-miR-315-5p effectively inhibits the translation of the EsFem-1c gene, influencing male development. An intriguing finding was that miRNA tcf-miR-307 could increase EsFem-1c expression by binding to the alternative splicing region with a length of 248 bp (ASR-248) in the 3'UTR sequence. The present research contributes to a better understanding of the molecular regulation mechanism of EsFem-1c and provides a resource for future studies of the miRNA-mediated regulation of sexual development and regulation in E. sinensis.

Patellar instability-induced bone loss in the femoral trochlea is associated with the activation of the JAK1/STAT3 signaling pathway in growing mice.

INTRODUCTION: Patellar instability (PI) at an early age is believed closely correlated with bone loss in the development of the femoral trochlea and can cause trochlear dysplasia. However, the molecular mechanism of PI-induced bone loss has not been established. The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway plays an important role in bone development by regulating the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL). The aim of this study was to explore the association of JAK1/STAT3 signaling to PI-induced subchondral bone loss in the femoral trochlea. METHODS: Four-week-old male C57BL/6 mice were randomly divided into two groups (n = 50/group). Mice in the experimental group underwent surgery to induce PI. Distal femurs were collected 2 and 4 weeks after surgery (n = 25 knees/each time point, each group). Microcomputed tomography and histological observations were performed to investigate the morphology of the femoral trochlea and changes in bone mass. qPCR, western blot, and immunohistochemistry analyses were performed to evaluate the expression of JAK1, STAT3, RANKL, and OPG in subchondral bone. A t test was performed for the statistical analysis; a P value < 0.05 was considered to be statistically significant. RESULTS: In the experimental group, subchondral bone loss in the femoral trochlea was observed two and four weeks after PI; morphological changes, such as a flatter trochlear groove and an increased sulcus angle, were observed in the femoral trochlea; qPCR, western blot, and immunohistochemistry analyses showed higher expression of JAK1, STAT3, and RANKL and lower expression of OPG (P < 0.05). CONCLUSION: PI-induced subchondral bone loss in the femoral trochlea and resulted in trochlear dysplasia in growing mice. This bone loss is associated with activation of the JAK1/STAT3 signaling pathway, which weakens the function of osteoblasts and stimulates both formation and function of osteoclasts.

Analyses of the Dmrt family in a decapod crab, Eriocheir sinensis uncover new facets on the evolution of DM domain genes.

DM domain genes are a group of transcription factors that are integral to sexual development and its evolution in metazoans. Their functions and regulatory mechanisms are not well understood in Malacostraca (crabs and crayfish) while these sex regulators have been widely identified in the past decade. In this study, the Dmrt family was investigated in the decapod crab, Eriocheir sinensis. We find that most members of the EsDmrt family begin to enrich around the juvenile 1 stage. In reproductive organs, EsDsx1, EsDsx2, EsiDMY and EsiDmrt1a highly express in the male-specific androgenic gland (AG), while EsDmrt-like, EsDsx-like, EsDmrt11E, and EsiDmrt1b show relatively high expression in testis. Also, we find the highly aberrant expression of EsiDMY and EsiDmrt1a in the chimeric AG, strongly indicating their function in AG development. Moreover, RNA interference of EsDsx1, EsiDMY, and EsiDmrt1a results in a significant decrease in transcription of the Insulin-like androgenic hormone (IAG), respectively. Our findings suggest that Dmrt genes in E. sinensis primarily function in male sexual differentiation, especially in AG development. Besides, this study identifies two unique groups of Dmrt genes in Malacostraca: Dsx and iDmrt1. In Malacostraca Dsx, we uncover a cryptic mutation in the eight zinc motif-specific residues, which were firmly believed to be invariant across the Dmrt family. This mutation sets the Malacostraca Dsx apart from all the other Dmrt genes and implies a different way of transcriptional regulation. Genes from the iDmrt1 group show phylogenetical limitation to the malacostracan species and underwent positive selection, suggesting their highly specialized gene function to this class. Based on these findings, we propose that Dsx and iDmrt1 in Malacostraca have developed unique transcriptional regulation mechanisms to facilitate AG development. We hope that this study would contribute to our understandings of sexual development in Malacostraca and provide new insights into the evolutionary history of the Dmrt family.

Identifying sex-differential gene expression in the antennal gland of the swimming crab by transcriptomic analysis.

The antennal glands (AnGs) are recognized as an important organ that functions in ion regulation and excretion in decapods. Previously, many studies had explored this organ at the biochemical, physiological, and ultrastructural levels but had few molecular resources. In this study, the transcriptomes of the male and female AnGs of Portunus trituberculatus were sequenced using RNA sequencing (RNA-Seq) technology. Genes involved in osmoregulation and organic/inorganic solute transport were identified. This suggests that AnGs might be involved in these physiological functions as versatile organs. A total of 469 differentially expressed genes (DEGs) were further identified between male and female transcriptomes and found to be male-biased. Enrichment analysis showed that females were enriched in amino acid metabolism and males were enriched in nucleic acid metabolism. These results suggested differences in possible metabolic patterns between males and females. Furthermore, two transcription factors related to reproduction, namely AF4/FMR2 family members Lilli (Lilli) and Virilizer (Vir), were identified in DEGs. Lilli was found to be specifically expressed in the male AnGs, whereas Vir showed high expression levels in the female AnGs. The expression of up-regulated metabolism and sexual development-related genes in three males and six females was verified by qRT-PCR and the pattern was found to be consistent with the transcriptome expression pattern. Our results suggest that although the AnG is a unified somatic tissue composed of individual cells, it still demonstrates distinct sex-specific expression patterns. These results provide foundational knowledge of the function and differences between male and female AnGs in P. trituberculatus.

New insight of variable lymphocyte receptor-likes in anti-bacteria activity from Eriocheir sinensis.

The Chinese mitten crab, Eriocheir sinensis, is a vital freshwater aquaculture species in China, however, is also facing various crab disease threats. In the present study, we identify three novel variable lymphocyte receptor-like (VLR-like) genes-VLR-like1, VLR-like3 and VLR-like4-from E. sinensis, which play vital roles in adaptive immune system of agnathan vertebrates. The bacterial challenge, bacterial binding and antibacterial-activity experiments were applied to study immune functions of VLR-likes, and the transcriptomic data from previous E. sinensis bacterial challenge experiments were analyzed to speculate the possible signaling pathway. VLR-like1 and VLR-like4 can respond to Staphylococcus aureus challenge and inhibit S. aureus specifically. VLR-like1 and VLR-like4 possess broad-spectrum bacteria-binding ability whereas VLR-like3 do not. VLR-likes in E. sinensis could associate with the Toll-like receptor (TLR) signaling pathway. The above results suggest that VLR-likes defend against bacteria invasion though exerting anti-bacteria activity, and probably connect with the TLR signaling pathway. Furthermore, studying the immune functions of these VLR-likes will provide a new insight into the disease control strategy of crustacean culture.

The antibacterial activity and antibacterial mechanism analyses of an LRR-IG protein in the Chinese mitten crab, Eriocheir sinensis.

Leucine-rich repeat and immunoglobulin domain containing protein (LRR-IG) family is an important class of immune molecules in invertebrates. Herein, a novel LRR-IG, named as EsLRR-IG5, was identified from Eriocheir sinensis. It contained typical structures of LRR-IG including an N-terminal LRR region and three IG domains. EsLRR-IG5 was ubiquitously expressed in all the tested tissues, and its transcriptional levels increased after being challenged with Staphylococcus aureus and Vibrio parahaemolyticus. Recombinant proteins of LRR and IG domains from the EsLRR-IG5 (named as rEsLRR5 and rEsIG5) were successfully obtained. rEsLRR5 and rEsIG5 could bind to both gram-positive bacteria and gram-negative bacteria as well as lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, rEsLRR5 and rEsIG5 exhibited antibacterial activities against V. parahaemolyticus and V. alginolyticus and displayed bacterial agglutination activities against S. aureus, Corynebacterium glutamicum, Micrococcus lysodeikticus, V. parahaemolyticus and V. alginolyticus. The scanning electron microscopy (SEM) observation revealed that the membrane integrity of V. parahaemolyticus and V. alginolyticus was destroyed by rEsLRR5 and rEsIG5, which may lead to the leakage of cell contents and death. This study provided clues for further studies on the immune defense mechanism mediated by LRR-IG in crustaceans and provided candidate antibacterial agents for prevention and control of diseases in aquaculture.

HSP40 mediated TLR-Dorsal-AMPs pathway in Portunus trituberculatus.

Heat shock protein 40 (HSP40) is a kind of molecular chaperone involved in various immune responses. However, the exact roles of HSP40 in immune defense against bacteria remain largely unclear. In this study, the activation function of a type Ⅰ HSP40 from Portunus trituberculatus (PtHSP40-Ⅰ) in the TLR pathway was investigated. The results showed that PtHSP40-Ⅰ can bind to lipopolysaccharide (LPS) and peptidoglycan (PGN). The PtHSP40-Ⅰ also exhibited binding activity toward the extracellular leucine-rich repeat (LRR) domain of Toll-like receptor (TLR). Moreover, the PtHSP40-Ⅰ could promote the transcription factor Dorsal translocated from cytoplasm into the nucleus in hemocytes and participated in regulating the expression of anti-lipopolysaccharide factor (ALF) and crustin. These findings provided insights into the activation mechanisms of TLR pathway mediated by HSP40 and offered basic theory of disease control in P. trituberculatus aquaculture.

Spätzle, a signaling molecule that interacts with pathogen-associated molecules and Toll-like receptor in Portunus trituberculatus.

Spätzle is a crucial ligand for Toll-like receptor (TLR) that triggers the activation of TLR signal pathway in insects. In this study, open reading frames (ORFs) of two spätzles were cloned from Portunus trituberculatus (PtSpz1 and PtSpz2). Both of PtSpzs contained the typical cystine-knot domain of spätzle. Tissue distribution analysis showed that both of PtSpzs were predominantly expressed in the gills. Transcriptional levels of the two PtSpzs in hemocytes and gill rapidly increased at 3 h and 6 h post Vibrio alginolyticus challenge, respectively. The two PtSpzs could bind to several pathogen-associated molecules including lipopolysaccharide (LPS), peptidoglycan (PGN) and envelope proteins of white spot syndrome virus (WSSV). Moreover, the two PtSpzs could directly interact with the extracellular leucine-rich repeats (LRR) domain of TLR. This study revealed that spätzle could interact with pathogen-associated molecules and TLR of host, which may be two important steps for spätzle to deliver signals into host cells.

New insights for the regulatory feedback loop between type 1 crustacean female sex hormone (CFSH-1) and insulin-like androgenic gland hormone (IAG) in the Chinese mitten crab (Eriocheir sinensis).

To clarify the hormone control on sex determination and differentiation, we studied the Chinese mitten crab, Eriocheir sinensis (Henri Milne Edwards, 1854), a species with importantly economic and ecological significance. The crustacean female sex hormone (CFSH) and the insulin-like androgenic gland hormone (IAG) have been found to be related to the sex determination and/or differentiation. CFSH-1 of E. sinensis (EsCFSH-1) encoded a 227 amino-acid protein including a signal peptide, a CFSH-precursor-related peptide, and a mature CFSH peptide. Normally, EsCFSH-1 was highly expressed in the eyestalk ganglion of adult female crabs, while the expression was declined in the intersex crabs (genetic females). The intersex crabs had the androgenic glands, and the expression level of EsIAG was close to that of male crabs. During the embryogenesis and larval development, the changes of EsCFSH-1 and EsIAG genes expression in male and female individuals were shown after the zoea IV stage. Next, we confirmed the existence of the regulatory feedback loop between EsCFSH-1 and EsIAG by RNA interference experiment. The feminization function of EsCFSH-1 was further verified by examining the morphological change of external reproductive organs after EsCFSH-1 knockdown. The findings of this study reveal that the regulatory interplay between CFSH and IAG might play a pivotal role in the process of sex determination and/or differentiation in decapod crustaceans.

Identification and functional characterization of C-type lectins and crustins provide new insights into the immune response of Portunus trituberculatus.

A meticulous understanding of the immune characteristics of aquaculture animals is the basis for developing precise disease prevention and control strategies. In this study, four novel C-type lectins (PtCTL-5, PtCTL-6, PtCTL-7 and PtCTL-8) including a single carbohydrate-recognition domain (CRD), and four novel crustins (Ptcrustin-1, Ptcrustin-2, Ptcrustin-3 and Ptcrustin-4) with a single whey acidic protein (WAP) domain were identified from the swimming crab Portunus trituberculatus. Tissue distribution analysis indicated that most of the target genes were predominantly expressed in the hepatopancreas in all examined tissues, except for Ptcrustin-1 which were mainly expressed in the gills. Our results showed that the eight genes displayed various transcriptional profiles across different tissues. In hemocytes, the PtCTL-7 responded quickly to Vibrio alginolyticus and exhibited much more strongly up-regulation than other three PtCTLs. The Ptcrustin-1 rapidly responded to V. alginolyticus within 3 h in all the three tested tissues. Furthermore, recombinant proteins of PtCTL-5 and PtCTL-8 were successfully obtained, and both of them displayed bacterial binding activities toward V. alginolyticus, V. harveyi and Staphylococcus aureus, and only showed antibacterial activity against V. harveyi. These findings provided new insights into the diverse immune response of P. trituberculatus and laid theoretical foundations for the development of precise disease prevention and control strategies in P. trituberculatus farming. Moreover, the specific anti-V. harveyi activities exhibited by rPtCTL-5 and rPtCTL-8 suggested their promising application prospects for controlling diseases caused by V. harveyi.

Anatomical Components Associated With Increased Tibial Tuberosity-Trochlear Groove Distance.

Background: Increased tibial tuberosity-trochlear groove (TT-TG) distance is an important indicator of medial tibial tubercle transfer in the surgical management of lateral patellar dislocation (LPD). Changes to TT-TG distance are determined by a combination of several anatomical factors. Purpose: To (1) determine the anatomical components related to increased TT-TG distance and (2) quantify the contribution of each to identify the most prominent component. Study Design: Case-control study; Level of evidence, 3. Methods: Included were 80 patients with recurrent LPD and 80 age- and body mass index-matched controls. The 2 groups were compared in TT-TG distance and its related anatomical components: tibial tubercle lateralization (TTL), trochlear groove medialization, femoral anteversion, tibiofemoral rotation (TFR), tibial torsion, and mechanical axis deviation (MAD). The Pearson correlation coefficient (r) was calculated to evaluate the association between increased TT-TG distance and its anatomical parameters, and factors that met the inclusion criteria of P < .05 and r ≥ 0.30 were analyzed via stepwise multivariable linear regression analysis to predict TT-TG distance. Results: The LPD and control groups differed significantly in TT-TG distance, TTL, TFR, and MAD (P < .001 for all). Increased TT-TG distance was significantly positively correlated with TTL (r = 0.376; P < .001), femoral anteversion (r = 0.166; P = .036), TFR (r = 0.574; P < .001), and MAD (r = 0.415; P < .001), and it was signficantly negatively correlated with trochlear groove medialization (r = -0.178; P = .024). The stepwise multivariable analysis revealed that higher TTL, excessive knee external rotation, and excessive knee valgus were statistically significant predictors of greater TT-TG distance (P < .001 for all). The standardized estimates that were used for evaluating the predictive values were larger for TFR compared with those for TTL and MAD. Conclusion: TTL, TFR, and MAD were the main independent anatomical components associated with increased TT-TG distance, with the most prominent component being TFR. The association of TT-TG distance to each component analyzed in our study may help guide surgical planning.

Full-Length Transcriptome Analysis Provides New Insights Into the Diversity of Immune-Related Genes in Portunus trituberculatus.

Generally, invertebrates were thought to solely rely on their non-specific innate immune system to fight against invading microorganisms. However, increasing studies have implied that the innate immune response of invertebrates displayed diversity and specificity owing to the hyper-variable immune molecules in organisms. In order to get an insight into the diversity of immune-related genes in Portunus trituberculatus, a full-length transcriptome analysis of several immune-related tissues (hemocytes, hepatopancreas and gills) in P. trituberculatus was performed and the diversity of several immune-related genes was analyzed. The full-length transcriptome analysis of P. trituberculatus was conducted using a combination of SMRT long-read sequencing and Illumina short-read sequencing. A total of 17,433 nonredundant full-length transcripts with average length of 2,271 bp and N50 length of 2,841 bp were obtained, among which 13,978 (80.18%) transcripts were annotated. Moreover, numerous transcript variants of various immune-related genes were identified, including pattern recognition receptors, antimicrobial peptides, heat shock proteins (HSPs), antioxidant enzymes and vital molecules in prophenoloxidase (proPO)-activating system. Based on the full-length transcriptome analysis, open reading frames (ORFs) of four C-type lectins (CTLs) were cloned, and tissue distributions showed that the four CTLs were ubiquitously expressed in all the tested tissues, and mainly expressed in hepatopancreas and gills. The transcription of the four CTLs significantly increased in several immune-related tissues (hemocytes, hepatopancreas and gills) of P. trituberculatus challenged with Vibrio alginolyticus and displayed different profiles. Moreover, the four CTLs displayed distinct bacterial binding and antibacterial activities. The recombinant protein PtCTL-1 (rPtCTL-1) and rPtCTL-3 displayed bacterial binding and antibacterial activities against all tested bacteria. rPtCTL-2 only showed bacterial binding and antibacterial activities against V. alginolyticus. No obvious bacterial binding or antibacterial activities for PtCTL-4 was observed against the tested bacteria. This study enriches the transcriptomic information on P. trituberculatus and provides new insights into the innate immune system of crustaceans. Additionally, our study provided candidates of antibiotic agents for the prevention and treatment of bacteriosis.

Alternative splicing derived invertebrate variable lymphocyte receptor displays diversity and specificity in immune system of crab Eriocheir sinensis.

Variable lymphocyte receptors (VLRs) play vital roles in adaptive immune system of agnathan vertebrate. In the present study, we first discover a novel VLR gene, VLR2, from an invertebrate, the Chinese mitten crab, Eriocheir sinensis. VLR2 has ten different isoforms formed via alternative splicing, which is different from that in agnathan vertebrate with the assembly of LRR modules. The longest isoform, VLR2-L, responds to Gram-positive bacteria Staphylococcus aureus challenge specifically, while shows no response to Gram-negative bacteria Vibrio parahaemolyticus challenge, confirmed by recombinant expression and bacterial binding experiments. Interestingly, VLR2s with short LRRs regions (VLR2-S8 and VLR2-S9) tend to bind to Gram-negative bacteria rather than Gram-positive bacteria. Antibacterial activity assay proves six isoforms of VLR2 have pluralistic antibacterial effects on bacteria which were never reported in invertebrate. These results suggest that the diversity and specificity of VLR2 resulted from alternative splicing and the length of the LRRs region. This pathogen-binding receptor diversity will lay the foundation for the study of immune priming. Furthermore, studying the immune function of VLR2 will provide a new insight into the disease control strategy of crustacean culture.

Genome-Wide Identification, Characterization, and Expression Analysis of DDE_Tnp_4 Family Genes in Eriocheir sinensis.

DDE transposase 4 (DDE_Tnp_4) family is a large endonuclease family involved in a wide variety of biological processes. However, little information is available about this family in crustaceans. In this study, we used HMMER to identify 39 DDE_Tnp_4 family genes in Eriocheir sinensis genome, and the genes were classified into four subfamilies according to phylogenetic analysis. Gene expansions occurred among E. sinensis genome, and synteny analysis revealed that some DDE_Tnp_4 family genes were caused by tandem duplication. In addition, the expression profiles of DDE_Tnp_4 family genes in E. sinensis indicated that subfamily I and II genes were up-regulated in response to acute high salinity and air exposure stress. E. sinensis is a kind of economical crustacean with strong tolerance to environmental stress. We confirmed the expansion of DDE_Tnp_4 family genes in E. sinensis and speculated that this expansion is associated with strong tolerance of E. sinensis. This study sheds light on characterizations and expression profiles of DDE_Tnp_4 family genes in E. sinensis and provides an integrated framework for further investigation on environmental adaptive functions of DDE_Tnp_4 family in crustaceans.