IL21 inhibits miR-361-5p to promote MAP3K9 and further aggravate the progression of shoulder arthritis.

OBJECTIVE: This research aimed to explore IL-21/miR-361-5p/MAP3K9 expression in shoulder arthritis and identify its regulatory pathways. METHODS: We established a rat shoulder arthritis model, then quantified IL21 and miR-361-5p in synovial fluid using ELISA and monitored the arthritis development. Additionally, IL21's effect on miR-361-5p levels in cultured human chondrocytes (HC-a) was assessed. Chondrocyte cell cycle status and apoptosis were measured via flow cytometry. Interactions between miR-361-5p and MAP3K9 were confirmed through dual-luciferase reporting and bioinformatic scrutiny. Protein levels of MAP3K9, p-ERK1/2, p-NF-κB, MMP1, and MMP9 were analyzed by Western blots. RESULTS: IL21 levels were elevated, while miR-361-5p was reduced in the synovial fluid from arthritic rats compared to healthy rats. IL21 was shown to suppress miR-361-5p in chondrocytes leading to hindered cell proliferation and increased apoptosis. Western blots indicated that miR-361-5p curbed MAP3K9 expression, reducing MMP activity by attenuating the ERK1/2/NF-κB pathway in chondrocytes. CONCLUSION: IL21 upregulation and miR-361-5p downregulation characterize shoulder arthritis, resulting in MAP3K9 overexpression. This chain of molecular events boosts MMP expression in chondrocytes and exacerbates the condition's progression.

Post-Subfunctionalization Functions of HIF-1αA and HIF-1αB in Cyprinid Fish: Fine-Tuning Mitophagy and Apoptosis Regulation Under Hypoxic Stress.

Hypoxia-inducible factor 1 (HIF-1) is a crucial transcriptional factor that can restore oxygen balance in the body by regulating multiple vital activities. Two HIF-1α copies were retained in cyprinid fish after experiencing a teleost-specific genome duplication. How the "divergent collaboration" of HIF-1αA and HIF-1αB proceeds in regulating mitophagy and apoptosis under hypoxic stress in cells of cyprinid fish remains unclear. In this study, zebrafish HIF-1αA/B expression plasmids were constructed and transfected into the epithelioma papulosum cyprini cells and were subjected to hypoxic stress. HIF-1αA induced apoptosis through promoting ROS generation and mitochondrial depolarization when cells were subjected to oxygen deficiency. Conversely, HIF-1αB was primarily responsible for mitophagy induction, prompting ATP production to mitigate apoptosis. HIF-1αA did not induce mitophagy in the mitochondria and lysosomes co-localization assay but it was involved in the regulation of different mitophagy pathways. Over-expression of HIF-1αA increased the expression of bnip3, fundc1, Beclin1, and foxo3, suggesting it has a dual role in mitochondrial autophagy and cell death. Each duplicated copy also experienced functional divergence and target shifting in the regulation of complexes in the mitochondrial electron transport chain (ETC). Our findings shed light on the post-subfunctionalization function of HIF-1αA and HIF-1αB in zebrafish to fine-tune regulation of mitophagy and apoptosis following hypoxia exposure.

Molecular characterization of a cation-dependent mannose-6-phosphate receptor gene in Crassostrea hongkongensis and its responsiveness in Vibrio alginolyticus infection.

The cation-dependent mannose-6-phosphate receptor (CD-M6PR) is a P-type lectin that plays a crucial role in lysosomal enzyme transport, bacterial resistance, and viral entry. In this study, we cloned and analyzed the ORF of the CD-M6PR gene from Crassostrea hongkongensis and named it ChCD-M6PR. We analyzed the nucleotide and amino acid sequence of ChCD-M6PR, its tissue expression pattern and immune response to Vibrio alginolyticus. Our results showed that the ORF of ChCD-M6PR was 801 bp long and encoded a protein of 266 amino acids with a signal peptide at the N-terminus, as well as Man-6-P_recep, ATG27 and transmembrane structural domains. Phylogenetic analysis indicated that Crassostrea hongkongensis shared the highest similarity with Crassostrea gigas in the terms of CD-M6PR. The ChCD-M6PR gene was found to be expressed in various tissues, with the highest expression observed in the hepatopancreas and the lowest in the hemocytes by the fluorescence quantitative PCR. Furthermore, the expression of ChCD-M6PR gene was significantly up-regulated for a short time in response to Vibrio alginolyticus infection in the gill and hemocytes, while it was down-regulated in the gonads. The expression patterns of ChCD-M6PR also varied in the other tissues. The 96 h cumulative mortality rate of Crassostrea hongkongensis infected with Vibrio alginolyticus after knockdown the ChCD-M6PR gene was significantly higher. Overall, our findings suggests that ChCD-M6PR plays a crucial role in the immune response of Crassostrea hongkongensis to Vibrio alginolyticus infection, and its tissue-specific expression patterns may be indicatitive of varied immune responses across tissues.

Pan-genome analysis of the Burkholderia gladioli PV. Cocovenenans reveal the extent of variation in the toxigenic gene cluster.

Burkholderia gladioli has been reported as the pathogen responsible for cases of foodborne illness in many countries. The poisonous bongkrekic acid (BA) produced by B. gladioli was linked to a gene cluster absent in non-pathogenic strains. The whole genome sequence of eight bacteria strains, which were screened from the collected 175 raw food and environmental samples, were assembled and analyzed to detect a significant association of 19 protein-coding genes with the pathogenic status. Except for the common BA synthesis-related gene, several other genes, including the toxin-antitoxin genes, were also absent in the non-pathogenic strains. The bacteria strains with the BA gene cluster were found to form a single cluster in the analysis of all B. gladioli genome assemblies for the variants in the gene cluster. Divergence of this cluster was detected in the analysis for both the flanking sequences and those of the whole genome level, which indicates its complex origin. Genome recombination was found to cause a precise sequence deletion in the gene cluster region, which was found to be predominant in the non-pathogenic strains indicating the possible effect of horizontal gene transfer. Our study provided new information and resources for understanding the evolution and divergence of the B. gladioli species.

Gender-Specific Metabolic Responses of Crassostrea hongkongensis to Infection with Vibrio harveyi and Lipopolysaccharide.

Gender differences in the hemocyte immune response of Hong Kong oyster Crassostrea hongkongensis to Vibrio harveyi and lipopolysaccharide (LPS) infection exist. To determine if a gender difference also exists, we use a 1H NMR-based metabolomics method to investigate responses in C. hongkongensis hepatopancreas tissues to V. harveyi and LPS infection. Both infections induced pronounced gender- and immune-specific metabolic responses in hepatopancreas tissues. Responses are mainly presented in changes in substances involved in energy metabolism (decreased glucose, ATP, and AMP in males and increased ATP and AMP in LPS-infected females), oxidative stress (decreased glutathione in males and decreased tryptophan and phenylalanine and increased choline and proline in LPS-infected females), tricarboxylic acid (TCA) cycle (decreased α-ketoglutarate acid and increased fumarate in LPS-infected males, and decreased fumarate in LPS-infected females), and osmotic regulation (decreased trigonelline and increased taurine in V. harveyi-infected males and decreased betaine in V. harveyi-infected females). Results suggest that post-spawning-phase male oysters have a more significant energy metabolic response and greater ability to cope with oxidative stress than female oysters. We propose that the impact of oyster gender should be taken into consideration in the aftermath of oyster farming or oyster disease in natural seas.

Mechanism of enrofloxacin-induced multidrug resistance in the pathogenic Vibrio harveyi from diseased abalones.

Vibrio harveyi infection had caused severe economic losses in aquaculture. A pathogenic V. harveyi strain had been successfully induced to be a multiple-resistant strain by enrofloxacin (EFX), then the mechanism of multidrug resistance was analyzed. It suggested that the minimum inhibitory concentration of EFX increased by 32-folds. Results of the Kirby-Bauer test showed that the inhibitory zone diameter was 25.3 mm for the sensitive strain (labeled as HL-S) and 8.5 mm for the resistant strain (labeled as HL-R). After 20 serial passages, even when the stress of EFX was removed, the resistance persisted. After induction of EFX, HL-R resisted to other fluoroquinolones, it even resisted to furazolidone and streptomycin, although it was sensitive to these antibiotics initially. Its sensitivity to rifampicin and doxycycline also decreased obviously. Results showed that 3522 differentially expressed genes were identified. Expression of the multidrugs efflux resistance-nodulation-cell division was significantly upregulated (164.61-folds) in HL-R. Other key genes connected with drug efflux were also upregulated significantly (p<0.05). Notably, recA encoded for recombination protein was upregulated significantly, lexA was downregulated significantly in HL-R. Research results showed that the efflux system and the save our souls system have played crucial roles during the development of multidrug resistance of V. harveyi.

Transcriptome Sequencing and Screening of Anthocyanin-Related Genes in the Leaves of Acer truncatum Bunge.

Acer truncatum Bunge is generally used as an ornamental tree because of its autumn leaves, although the viewing period is short-approximately 7-15 days. Color improvement of ornamental trees has consistently been an important research topic because color partially determines the value of the commodity; however, a lack of genomic data have limited the progress of molecular breeding research in this area. The purposes of this study were to obtain a transcriptome database for A. truncatum, screen anthocyanin biosynthesis-related genes, and reveal the mechanisms underlying leaf color transformation to provide a basis for increasing the viewing period or breeding cultivars that display red leaves throughout the growing season via gene regulation. In this study, although the use of an Illumina HiSeq 2000 platform and systematic bioinformatics analysis using both young and mature leaves as experimental materials, 233,912,882 clean reads were generated and 121,287 unique transcripts were retrieved. We selected 16 color-related genes (from the transcriptome results) for qRT-PCR to validate the results, and the expression trends of the selected genes were largely consistent with the transcriptome analysis results, with a consistency of 0.875. According to the results of the transcriptome analysis, the validation, and previous studies, we obtained sequences of genes related to anthocyanins, including CHS, CHI, ANS, UFGT, UGT75c1, DFR, BZ1, F3H, F3'H, LAR, ANR, FLS, and those of several transcription factors, including MYB1, BHLH, and WD40. Verifying specific regulation by one or several of these genes in the control of leaf color requires further research. The acquisition of transcriptomic information, especially information concerning anthocyanin biosynthesis-related genes and their base sequences, can provide a theoretical basis for the study of the molecular mechanisms determining changes in leaf color in Acer and is of great importance to the breeding of new cultivars.

Transcriptome sequencing and flavonoid metabolism analysis in the leaves of three different cultivars of Acer truncatum.

Young and mature leaves of three Acer truncatum varieties with different leaf colors were examined. Transcriptome sequencing and flavonoid metabolism were used to analyze the differential gene expression associated with different leaf colors and growth stages and the relationships between gene expression and flavonoid and anthocyanin contents to improve ornamental value and develop flavonoid-rich A. truncatum. Kyoto Encyclopedia of Genes and Genomes database annotation of differentially expressed genes indicated that the following genes were related to flavonoid synthesis: phenylpropanoid biosynthesis genes (PAL, C4H, 4CL and CHS), flavonoid biosynthesis genes (E2.1.1.104, CHI, FLS, F3'5'H and ANR), anthocyanin biosynthesis genes (ANS, DFR, HCT, BZ1, GT1, and UGT79B1), isoflavonoid biosynthesis genes (HIDH and CYP81E17), and their transcriptional regulator (MYB). A total of 234 types of flavonoids were detected. The types and contents of anthocyanins in the red-leaf varieties 'Hong Jingling' and 'Caidie Fanfei' were significantly higher than those in the green leaf cultivar 'Lv Baoshi', especially morning glory 3-O-glucoside, delphinidin 3-O-glucoside, and pelargonium-3-O-glucoside, which were not detected in 'Lv Baoshi'. Combined omics analysis showed that downregulated expression of C4H, CHS and F3'5'H and upregulated expression of FLS reduced the supply of raw materials for anthocyanin synthesis, and downstream ANR upregulation converted anthocyanins to procyanidins, increasing the total flavonoid content. F3'5'H expression was downregulated in the leaves of each variety with development, resulting in the accumulation of catechins and the gradual greening of the leaves. F3'5'H was significantly depleted in the young leaves of 'Hong Jingling' and 'Caidie Fanfei' compared with the young leaves of 'Lv Baoshi', while ANS and BZ1 were enriched significantly. It is concluded that F3'5'H, BZ1, and ANS are the key genes needed for breeding red A. truncatum and that ANR is the key gene needed for breeding varieties with a high flavonoids contens. These results may facilitate genetic modification or selection for further improvement of the ornamental qualities and flavonoid content of A. truncatum.

Middle to late Holocene plant cover variation in relation to climate, fire, and human activity in the Songnen grasslands of northeastern China.

Introduction: For future vegetation projections and conservation planning in grassland ecosystems, accurate estimates of past plant cover changes in grassland composition and their responses to the various driving factors are essential. This study quantitatively reconstructs the past regional plant cover in the Songnen grasslands (northeastern China) and explores the relative importance of climate, fire, and human activity on vegetation dynamics. Methods: For this purpose, the Regional Estimates of Vegetation Abundance from Large Sites (REVEALS) model is applied to three pollen records from two areas, two in the center of the Songnen grasslands and one located in an area marginal to the grasslands. Results: Results from the most reliable REVEALS scenarios show that from the mid-Holocene, steppe (mean cover 40.6%) and dry steppe (mean cover 54.2%) alternately dominated the central part of the Songnen grasslands while the marginal grasslands were mainly characterized by alternating broadleaved forests (mean cover 26.3%), coniferous forests (mean cover 41.9%) and dry steppes (mean cover 30.1%). Discussion: By comparing the plant cover results with previous published regional climate, fire and human activity records, the results show that long term vegetation dynamics were mainly driven by East Asia Summer Monsoon (EASM) and the related precipitation variations, but was also affected by fire frequency and human activity. Moreover, vegetation evolution was sensitive to abrupt cooling events including the 4.2 ka BP and stacked ice-rafted debris (IRD) events; the change from steppe to dry steppe, for example, was driven by these abrupt climate changes. Fire events can alter the original vegetation stability allowing the vegetation to respond rapidly to climate changes while human activity merely has limited influence on vegetation changes.

Highly sensitive non-enzymatic hydrogen peroxide monitoring platform based on nanoporous gold via a modified solid-phase reaction method.

In this work, nanoporous gold (NPG) fabricated using a modified solid-phase reaction method was developed as an electrocatalyst for the nonenzymatic detection of hydrogen peroxide (H2O2). The NPG morphology and structure were characterized by scanning electron microscopy and high-resolution transmission electron microscopy. The fabricated NPG exhibited a nanoporous framework with numerous structural defects. The NPG-based amperometric H2O2 sensor had a good selectivity, reproducibility, and low detection limit (0.3 µM) under near physiological conditions (pH = 7.4). The sensitivities of this sensor over concentration ranges of 0.002-5 mM and 5-37.5 mM were 159 and 64 µA mM-1 cm-2, respectively. These results indicate that the developed NPG is a promising material for the electrochemical sensing of H2O2.

Growth of c-plane and m-plane aluminium-doped zinc oxide thin films: epitaxy on flexible substrates with cubic-structure seeds.

Manufacturing high-quality zinc oxide (ZnO) devices demands control of the orientation of ZnO materials due to the spontaneous and piezoelectric polarity perpendicular to the c-plane. However, flexible electronic and optoelectronic devices are mostly built on polymers or glass substrates which lack suitable epitaxy seeds for the orientation control. Applying cubic-structure seeds, it was possible to fabricate polar c-plane and nonpolar m-plane aluminium-doped zinc oxide (AZO) films epitaxially on flexible Hastelloy substrates through minimizing the lattice mismatch. The growth is predicted of c-plane and m-plane AZO on cubic buffers with lattice parameters of 3.94-4.63 Šand 5.20-5.60 Å, respectively. The ∼80 nm-thick m-plane AZO film has a resistivity of ∼11.43 ±â€…0.01 × 10-4 Ω cm, while the c-plane AZO film shows a resistivity of ∼2.68 ±â€…0.02 × 10-4 Ω cm comparable to commercial indium tin oxide films. An abnormally higher carrier concentration in the c-plane than in the m-plane AZO film results from the electrical polarity along the c-axis. The resistivity of the c-plane AZO film drops to the order of 10-5 Ω cm at 500 K owing to the semiconducting behaviour. Epitaxial AZO films with low resistivities and controllable orientations on flexible substrates offer optimal transparent electrodes and epitaxy seeds for high-performance flexible ZnO devices.

In situ hybridization revealed wide distribution of Haliotid herpesvirus 1 in infected small abalone, Haliotis diversicolor supertexta.

Ganglioneuritis was the primary pathologic change in infected abalone associated with Haliotid herpesvirus 1 (HaHV-1) infection, which eventually became known as abalone viral ganglioneuritis (AVG). However, the distribution of HaHV-1 in the other tissues and organs of infected abalone has not been systemically investigated. In the present study, the distribution of HaHV-1-CN2003 variant in different organs of small abalone, Haliotis diversicolor supertexta, collected at seven different time points post experimental infection, was investigated with histopathological examination and in situ hybridization (ISH) of HaHV-1 DNA. ISH signals were first observed in pedal ganglia at 48 h post injection, and were consistently observed in this tissue of challenged abalone. At the same time, increased cellularity accompanied by ISH signals was observed in some peripheral ganglia of mantle and kidney. At the end of infection period, lesions and co-localized ISH signals in infiltrated cells were detected occasionally in the mantle and hepatopancreas. Transmission electron microscope analysis revealed the presence of herpes-like viral particles in haemocyte nuclei of infected abalone. Our results indicated that, although HaHV-1-CN2003 was primarily neurotropic, it could infect other tissues including haemocytes.

Control of biofouling on pearl oysters Pinctada imbricata using wax and Chinese herbs.

Biofouling poses severe challenges to pearl oyster Pinctada imbricata culture in China, and controlling it is both labor- and capital-intensive. The antifouling properties of wax, and wax mixed with Chinese herbs, sprayed onto pearl oyster shell surfaces during peak biofouling seasons were evaluated. Pearl oysters coated with three wax treatments (plain wax, Chinaberry seed extract, Chinese honeylocust fruit extract) and a control (no treatment), were cultured in nets for up to 60 days. Mortality rate, fouling organism and pearl-oyster weights, and shell height are reported for individual oysters on each of six sampling dates. With the exception of oysters submerged for 12 days, all oysters were significantly affected by treatment type and submersion duration. Fouling weight increased more rapidly over time in the control-treatment oysters. Wax-based coatings deterred fouling-organism settlement on oysters for at least 2 months during the intensive fouling season, reducing mortality and not adversely effecting growth.

Utilization of recombinase polymerase amplification combined with a lateral flow strip for detection of Perkinsus beihaiensis in the oyster Crassostrea hongkongensis.

BACKGROUND: Perkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37-42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1-10 target DNA copies in less than 20 minutes. METHODS: We report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye. RESULTS: The LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20-25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field. CONCLUSIONS: Our LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.

Beyond dealloying: development of nanoporous gold via metal-induced crystallization and its electrochemical properties.

Nanoporous metals (NPMs) possess a number of intriguing properties that result in NPMs being an important family of nanomaterials for many advanced applications. However, the methods of preparing NPMs are relatively complicated and have many limitations, which have hindered the commercial application of NPMs thus far. By introducing metal-induced crystallization, a solid-phase reaction method for preparing NPMs was developed in this study, which is highly efficient and environmentally friendly. The microstructure of the prepared nanoporous gold (NPG) was characterized on an atomic scale by scanning electron microscopy and high-resolution transmission electron microscopy. The results confirmed that the solid-phase reaction method is an effective alternative means of preparing highly pure NPG. The results of electrochemical tests demonstrated that thus-prepared NPG possesses higher electrocatalytic activity than other types of gold electrodes toward oxygen reduction in alkaline media. The combination of a simple preparation process and higher activity suggests that the developed method may promote the future use of NPG in new energy applications, such as fuel cells and metal-air batteries.

First report of black-heart disease in Kumamoto oyster Crassostrea sikamea spat caused by Polydora lingshuiensis in China.

A black-heart disease caused by polydorid infestation is reported for the first time in Kumamoto oyster Crassostrea sikamea Amemiya, 1928 spat in a pond at Beihai city, Guangxi province, China, with a prevalence of 100% and a cumulative mortality rate of 50% within 2 mo. In heavily infected oyster spat, blisters extended toward the center of the inner shell surface, around the adductor muscle scar area to form a large black area occupying approximately 50% of the area of the inner shell surface. Morphological analysis identified the pathogen as Polydora lingshuiensis Ye et al., 2015, which was reconfirmed by comparison of its corresponding 18S rRNA and mitochondrial CO1 gene sequences with those in the GenBank database. The mean abundance of mud blisters was significantly higher in live spat than in dead spat, suggesting that P. lingshuiensis preferentially infests live oyster spat. Additionally, P. lingshuiensis larvae were detected in the inlet near the dam, which suggests that the source of P. lingshuiensis larvae infecting the spat may be larvae entering the ponds through the water current from the sea.

Development of a Blocking Primer to Inhibit the PCR Amplification of the 18S rDNA Sequences of Litopenaeus vannamei and Its Efficacy in Crassostrea hongkongensis.

Diversity analyses of the eukaryotic microorganisms in the gut of marine animals is hampered by the presence of host DNA in the samples. PCR amplification of rRNA genes of eukaryotic microorganisms is inefficient with universal primers targeting 18S rRNA gene when the host DNA is dominant. In this study, we designed several blocking primers to inhibit PCR amplification of rRNA genes of the shrimp Litopenaeus vannamei, and tested their efficacy on the oyster Crassostrea hongkongensis. We first compared the intensity of PCR product bands obtained with and without the blocking primers. Then, one primer was selected for further verification using high-throughput sequencing. Our results showed that X-BP2-DPO was the most effective blocking primer in suppressing the host 18S amplification compared to nine other candidates. The inhibition rate was 99% for the amplification of shrimp rDNA, and 17% for the amplification of oyster rDNA. The concentration of the blocking primer in the PCR mixture was an important factor to be considered in the experimental design. The development of blocking primers provided a valid method to study the composition and characteristics of eukaryotic microorganisms in shrimp gut for a better understanding of its diets.

Molecular characterization and immune analysis of a defensin from small abalone, Haliotis diversicolor.

As one of antimicrobial peptides (AMPs), defensins are involved in invertebrate innate immunity against invading pathogens. In this study, a member of the invertebrate defensins was cloned and characterized from the small abalone Haliotis diversicolor, designated HdDef-2. The HdDef-2 cDNA contained a 201 bp open reading frame encoding 66 amino acids including a signal peptide of 18 amino acids and a mature peptide of 48 amino acids. The mature peptide of HdDef-2 possessed similar features to other AMPs, such as lower molecular mass, net positive charge (+1), and a high hydrophobic residue ratio (45%). In addition, six cysteines in the mature peptide were arranged in the pattern C-X16-C-X3-C-X9-C-X4-C-X1-C and stabilized the α-helix/ß-sheet motif (CSαß) with three disulfide bonds (C1-C4, C2-C5 and C3-C6) in the predicted tertiary structure. Moreover, the similar three-dimensional structure to Anopheles gambiae defensin and a phylogenetic analysis suggest that HdDef-2 may be a new member of the arthropod defensin family. Quantitative real-time PCR analysis revealed that HdDef-2 transcripts were constitutively expressed in the mantle, gill, hepatopancreas, and foot, with the highest level in the hepatopancreas. It was observed that HdDef-2 transcripts were significantly induced in the hepatopancreas after infection by Vibrio harveyi. These results indicate that HdDef-2 may be involved in the immune response against invading pathogenic bacteria, but future work is needed to verify its antimicrobial activity in protein level and elucidate the underlying mechanisms.

Gene expression and phenoloxidase activities of hemocyanin isoforms in response to pathogen infections in abalone Haliotis diversicolor.

Hemocyanins (Hc), the main protein components of hemolymph in invertebrates, are not only involved in oxygen transport but also linked to non-specific immune responses. In this study, we used abalone (Haliotis diversicolor) Hc to study the basis of its diversified functions through gene, protein, peptides, and phenoloxidase (PO) activity levels. Three complete hemocyanin gene (HdH) sequences were cloned for the first time. By comparing the copies and location of HdH between abalone and other mollusks, we propose that Hc gene duplication and linkage is likely to be common during the evolution of mollusk respiratory proteins. We further demonstrate that all three genes could be expressed in abalone, with expression varying based on the developmental stages, tissue types, and different pathogen infections. However, HdH1 and HdH2 appear to be synthesized by the same cells by fluorescence in situ hybridization. Furthermore, the PO activity of HdH can be induced by trypsin, urea, and SDS in vitro. Viral infection can stimulate its PO activity in vivo by cleaving the protein into fragments. Consequently, we present a comprehensive study of abalone hemocyanin, providing important evidence for an in-depth understanding of the physiological and immune functions of Hc in mollusks.

Susceptibility of two abalone species, Haliotis diversicolor supertexta and Haliotis discus hannai, to Haliotid herpesvirus 1 infection.

Abalone viral ganglioneuritis (AVG), caused by Haliotid herpesvirus-1 (HaHV-1) infection, has been reported as the main cause of mortality and heavy losses of wild and cultivated abalone in Taiwan and Australia since 2003. HaHV-1 DNA has also been reported in diseased abalone collected in early 2000s in China. However, no data is available about the susceptibility, disease process and pathological changes of HaHV-1 infection in the primary cultivated abalone species in China. In the present study, two cultivated abalone species, Haliotis diversicolor supertexta and Haliotis discus hannai, were challenged with HaHV-1-CN2003 collected in 2003 in China using three different methods. Results showed that H. diversicolor supertexta was highly susceptible to HaHV-1-CN2003 infection and suffered acute mortality using all three challenge methods. H. discus hannai was not susceptible to the viral infection. Histopathology combined with transmission electron microscopy and quantitative PCR analysis revealed that the tropism of HaHV-1-CN2003 includes both neural tissue and haemocytes.